EP4257156A2 - Conjugués de médicament à nanoparticules ciblées de récepteur de folate et leurs utilisations - Google Patents

Conjugués de médicament à nanoparticules ciblées de récepteur de folate et leurs utilisations Download PDF

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Publication number
EP4257156A2
EP4257156A2 EP23180783.5A EP23180783A EP4257156A2 EP 4257156 A2 EP4257156 A2 EP 4257156A2 EP 23180783 A EP23180783 A EP 23180783A EP 4257156 A2 EP4257156 A2 EP 4257156A2
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EP
European Patent Office
Prior art keywords
ndc
cancer
nanoparticle
linker
exatecan
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Pending
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EP23180783.5A
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German (de)
English (en)
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EP4257156A3 (fr
Inventor
Kai Ma
Aranapakam M. Venkatesan
Feng Chen
Fei Wu
Melik Ziya Türker
Thomas Courtney GARDINIER II
Geno J. GERMANO JR.
Francis Y. F. Lee
Gregory Paul Adams
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Elucida Oncology Inc
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Elucida Oncology Inc
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Publication of EP4257156A2 publication Critical patent/EP4257156A2/fr
Publication of EP4257156A3 publication Critical patent/EP4257156A3/fr
Pending legal-status Critical Current

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Definitions

  • ADCs Antibody drug conjugates
  • ADCs are a popular platform for targeted drug delivery, which typically feature a highly toxic drug substance covalently attached to a monoclonal antibody that can target cancer, wherein the toxic drug substance is released upon targeting of the cancer.
  • ADCs are a popular platform for targeted drug delivery, which typically feature a highly toxic drug substance covalently attached to a monoclonal antibody that can target cancer, wherein the toxic drug substance is released upon targeting of the cancer.
  • C'Dots can be conjugated with epidermal growth factor receptor inhibitors, e.g., gefitinib, which is a cancer-targeted agent that inhibits cancer growth ( WO 2015/183882 A1 ).
  • epidermal growth factor receptor inhibitors e.g., gefitinib
  • MOA mechanism of action
  • EGFR inhibitors requires active binding to the epidermal growth factor receptor, so a continuous high concentration of the payload in the targeted cancer cell is required to effectively inhibit cancer cell proliferation. This type of MOA is generally not compatible with the fast blood circulation half-life of C'Dots.
  • FR ⁇ Folate receptor alpha
  • FOLR1 Folate receptor alpha
  • FR Folate receptor
  • folate receptors of which there are four glycopeptide members (FR alpha [FOLR1], FR beta [FOLR2], FR gamma [FOLR3], and FR delta [FOLR4]).
  • the alpha isoform (FR alpha or FR ⁇ ) is a glycosylphosphatidylinositol (GPI)-anchored membrane protein with high affinity for binding and transporting the active form of folate, 5-methyltetrahydrofolate (5MTF).
  • GPI glycosylphosphatidylinositol
  • the alpha isoform has been reported to be over-expressed in certain solid tumors, for example, in ovarian cancer, fallopian tube cancer, primary peritoneum cancer, uterus cancer, kidney cancer, lung cancer, brain cancer, gastrointestinal cancer, and breast carcinomas.
  • the alpha isoform is also over-expressed in certain hematological malignancies, which can be exploited for treatment of these malignancies, e.g., for treatment of acute myeloid lymphoma (AML), including pediatric AML.
  • AML acute myeloid lymphoma
  • beta isoform is overexpressed in certain cancers, e.g., hematological malignancies such as acute myeloid leukemia (AML) and chronic myelogenous leukemia (CML), providing the opportunity to develop targeted therapies for these cancers.
  • AML acute myeloid leukemia
  • CML chronic myelogenous leukemia
  • NDC nanoparticle-drug conjugate
  • a silica nanoparticle, and polyethylene glycol (PEG) covalently bonded to the surface of the nanoparticle comprising: (a) a silica nanoparticle, and polyethylene glycol (PEG) covalently bonded to the surface of the nanoparticle; (b) a targeting ligand comprising folic acid, or a derivative or salt thereof, wherein the targeting ligand is attached to the nanoparticle directly or indirectly through a spacer group; and (c) a linker-payload conjugate, wherein: (i) the payload is exatecan; (ii) the linker-payload conjugate is attached to the nanoparticle directly or indirectly through a spacer group; (iii) the linker is a protease-cleavable linker; and (iv) the exatecan is released upon cleavage of the linker.
  • PEG polyethylene glycol
  • NDCs nanoparticle-drug-conjugates
  • a nanoparticle that comprises a silica-based core and a silica shell surrounding at least a portion of the core; polyethylene glycol (PEG) covalently bonded to the surface of the nanoparticle, and a fluorescent compound covalently encapsulated within the core of the nanoparticle;
  • PEG polyethylene glycol
  • a targeting ligand that binds to folate receptor (FR), wherein the targeting ligand comprises folic acid, or a folate receptor binding derivative thereof, and wherein the targeting ligand is attached to the nanoparticle directly or indirectly through a spacer group;
  • a linker-payload conjugate wherein the payload is a cytotoxic agent; wherein the linker-payload conjugate is attached to the nanoparticle directly or indirectly thorough a spacer group; wherein the cytotoxic agent is released upon cleavage of the linker; wherein the linker in the linker-payload
  • the average nanoparticle to payload ratio may range from 1 to 80, such as from 1 to 21 (e.g., 1 to 13, or 1 to 12) and the average nanoparticle to targeting ligand ratio may range from 1 to 50, such as from 1 to 25 (e.g., 1 to 11).
  • the NDCs of the present disclosure may have an average diameter of between about 1 nm and about 10 nm, e.g., between about 5 nm and about 8 nm, between about 3 nm and about 8 nm, or between about 3 nm and about 6 nm.
  • the NDCs of the present disclosure may comprise any suitable dye or detectable compound, such as a fluorescent compound.
  • the fluorescent compound may be Cy5.
  • the fluorescent compound may be encapsulated within the nanoparticle (e.g., covalently linked to the silica core).
  • the NDCs of the present disclosure can comprise a targeting ligand that binds to a folate receptor (FR).
  • the targeting ligand may comprise folic acid or a derivative thereof.
  • folic acid may encompass an amide or an ester of folic acid, e.g., folic acid may be conjugated to the nanoparticle (or spacer group) at its carboxyl terminus via an amide or ester bond.
  • folic acid may refer to the folic acid amide present in the exemplary NDC illustrated in FIG. 1 .
  • the NDCs of the present disclosure may comprise structure (S-1): wherein Payload comprises exatecan; Linker comprises a protease-cleavable linker; and the silicon atom is a part of the nanoparticle.
  • the NDCs of the present disclosure may comprise structure (S-1a): wherein the silicon atom is a part of the nanoparticle.
  • the NDCs of the present disclosure may comprise Structure (S-2) : wherein Targeting Ligand is folic acid, or a folate receptor binding derivative thereof, and the silicon atom is a part of the nanoparticle.
  • the NDCs of the present disclosure may comprise structure (S-2a) : wherein the silicon atom is a part of the nanoparticle.
  • the NDCs of the present disclosure may comprise a combination of Structures (S-1) and (S-2).
  • the NDCs may comprise both Structure (S-1a) and Structure (S-2a) , e.g., as depicted in FIG. 1 .
  • Structures S-1, S-1a, S-2, or S-2a may be present in the NDC at any desired ratio, e.g., at a ratio disclosed herein.
  • the disclosure also relates to NDCs comprising a nanoparticle that comprises a silica-based core and a silica shell surrounding at least a portion of the core; polyethylene glycol (PEG) covalently bonded to the surface of the nanoparticle; a fluorescent compound covalently encapsulated within the core of the nanoparticle; a targeting ligand, wherein the targeting ligand is folic acid; a linker-payload conjugate, wherein the linker-payload conjugate is a protease cleavable linker that is capable of undergoing hydrolysis at a C-terminal end upon protease binding thereby releasing the payload from the nanoparticle, wherein the protease comprises a serine protease or a cysteine protease, wherein the payload in the linker-payload conjugate is exatecan, or an analog of exatecan; and wherein the fluorescent compound is Cy5.
  • PEG polyethylene glycol
  • the disclosure also relates to NDCs comprising a nanoparticle that comprises a silica-based core and a silica shell surrounding at least a portion of the core; polyethylene glycol (PEG) covalently bonded to the surface of the nanoparticle; a Cy5 dye covalently encapsulated within the core of the nanoparticle; a targeting ligand that binds to folate receptor, wherein the targeting ligand is folic acid, and wherein the targeting ligand is attached to the nanoparticle indirectly through a spacer group; a linker-payload conjugate, wherein the linker-payload conjugate is attached to the nanoparticle indirectly through a spacer group, wherein the linker-payload conjugate comprises a compound comprising the structure ; and wherein the NDC has an average diameter between about 1 nm and about 10 nm (e.g., between about 1 and about 6 nm).
  • PEG polyethylene glycol
  • NDC nanoparticle drug conjugates
  • a silica nanoparticle that comprises a silica-based core and a silica shell surrounding at least a portion of the core; and polyethylene glycol (PEG) covalently bonded to the surface of the nanoparticle;
  • PEG polyethylene glycol
  • an exatecan-linker moiety comprising the structure of Formula (NP-3): (NP-3), wherein x is 4 and y is 9;
  • a targeting ligand moiety comprising the structure of Formula (NP-2) (NP-2), wherein x is 4 and y is 3, and wherein the exatecan-linker moiety and the targeting ligand moiety are each conjugated to a surface of the nanoparticle.
  • the NDC may comprise a fluorescent dye (e.g., Cy5) covalently encapsulated within the core of the nanoparticle.
  • This disclosure also provides a method of treating a folate receptor (FR)-expressing cancer (e.g., a folate receptor (FR)-expressing tumor), comprising administering to a subject in need thereof an effective amount of an NDC described herein.
  • the method may include administration of NDCs to the subject in need thereof intravenously.
  • the subject may have a cancer selected from the group consisting of ovarian cancer, endometrial cancer, fallopian tube cancer, cervical cancer, breast cancer (including, e.g., HER2+ breast cancer, HR+ breast cancer, HR- breast cancer, and triple-negative breast cancer), lung cancer (e.g., non-small cell lung cancer (NSCLC), mesothelioma, uterine cancer, gastrointestinal cancer (e.g., esophageal cancer, colon cancer, rectal cancer, and stomach cancer), pancreatic cancer, bladder cancer, kidney cancer, liver cancer, head and neck cancer, brain cancer, thyroid cancer, skin cancer, prostate cancer, testicular cancer, acute myeloid leukemia (AML, e.g., pediatric AML), and chronic myelogenous leukemia (CML).
  • NSCLC non-small cell lung cancer
  • mesothelioma uterine cancer
  • gastrointestinal cancer e.g., esophageal cancer, colon cancer, rectal cancer, and stomach
  • the NDCs of the present disclosure may also be used for targeting tumor associated macrophages, which may be used as a means to modify the immune status of a tumor in a subject.
  • the NDCs of the present disclosure may be used in a method of treating an advanced, recurrent, or refractory solid tumor.
  • an NDC for treating a folate receptor (FR)-expressing cancer (e.g., a folate receptor (FR)-expressing tumor).
  • the use may include administration of NDCs intravenously to the subject in need thereof.
  • the subject may have a cancer selected from the group consisting of ovarian cancer, endometrial cancer, fallopian tube cancer, cervical cancer, breast cancer (including, e.g., HER2+ breast cancer, HR+ breast cancer, HR- breast cancer, and triple-negative breast cancer), lung cancer (e.g., non-small cell lung cancer (NSCLC), mesothelioma, uterine cancer, gastrointestinal cancer (e.g., esophageal cancer, colon cancer, rectal cancer, and stomach cancer), pancreatic cancer, bladder cancer, kidney cancer, liver cancer, head and neck cancer, brain cancer, thyroid cancer, skin cancer, prostate cancer, testicular cancer, acute myeloid leukemia (AML, e.g., pediatric AML), and chronic
  • NDCs for use in the manufacture of a medicament for treating a folate receptor (FR)-expressing cancer (e.g., a folate receptor (FR)-expressing tumor).
  • FR folate receptor
  • the use in the manufacture of a medicament may include administration of NDCs intravenously to the subject in need thereof.
  • the use in the manufacture of a medicament may include administration of NDCs to a subject, wherein the subject has a cancer selected from the group consisting of ovarian cancer, endometrial cancer, fallopian tube cancer, cervical cancer, breast cancer (including, e.g., HER2+ breast cancer, HR+ breast cancer, HR-breast cancer, and triple-negative breast cancer), lung cancer (e.g., non-small cell lung cancer (NSCLC), mesothelioma, uterine cancer, gastrointestinal cancer (e.g., esophageal cancer, colon cancer, rectal cancer, and stomach cancer), pancreatic cancer, bladder cancer, kidney cancer, liver cancer, head and neck cancer, brain cancer, thyroid cancer, skin cancer, prostate cancer, testicular cancer, acute myeloid leukemia (AML, e.g., pediatric AML), and chronic myelogenous leukemia (CML).
  • the NDCs of the present disclosure may be used in the manufacture of a medicament for treating an advanced, recurrent
  • This disclosure also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an NDC and a pharmaceutically acceptable excipient.
  • the pharmaceutical compositions disclosed herein may be used for treating a folate receptor (FR)-expressing cancer (e.g., a folate receptor (FR)-expressing tumor).
  • FR folate receptor
  • FR folate receptor
  • nanoparticle drug conjugates which comprise a nanoparticle (e.g., a silica nanoparticle, such as a multi-modal silica-based nanoparticle) that allows conjugation to targeting ligands and to cytotoxic payloads, for detection, prevention, monitoring, and/or treatment of a disease, such as cancer.
  • a nanoparticle e.g., a silica nanoparticle, such as a multi-modal silica-based nanoparticle
  • cytotoxic payloads for detection, prevention, monitoring, and/or treatment of a disease, such as cancer.
  • compositions and methods of using a nanoparticle drug conjugate comprising: a nanoparticle; a targeting ligand that binds to a folate receptor (e.g., folic acid, or a derivative or salt thereof), and a linker-payload conjugate, that may comprise exatecan and a protease-cleavable linker.
  • NDC nanoparticle drug conjugate
  • the conjugation of both targeting ligands and linker-drug conjugates to the nanoparticle can be achieved via a highly efficient "click chemistry" reaction, which is fast, simple to perform, versatile, and results in high product yields.
  • the payload may be a cytotoxic agent comprising exatecan, or a salt or analog thereof, that is attached to the nanoparticle via a cleavable linker group.
  • the cleavable linker group can be cleaved when the NDC is internalized in a cancer cell (e.g., in a tumor cell), such as in the endosome or lysosomal compartment of the cell, causing release of the active cytotoxic agent from the NDCs.
  • the cleavage may be catalyzed by a protease (e.g., cathepsin B).
  • the NDCs disclosed herein provide an optimal platform for drug delivery, due in part to their physical properties.
  • the NDCs comprise nanoparticles that are ultrasmall in diameter (e.g., with average diameter between about 1 nm and about 10 nm, such as between about 5 nm and about 8 nm) and benefit from enhanced permeability and retention (EPR) effects in tumor microenvironments, while retaining desired clearance and pharmacokinetic properties.
  • EPR enhanced permeability and retention
  • the NDCs described herein have certain advantages over other drug delivery platforms (e.g., ADCs such as FR-targeted ADCs, and FR-targeted small molecule drugs (e.g., chemotherapeutics)).
  • ADCs such as FR-targeted ADCs, and FR-targeted small molecule drugs (e.g., chemotherapeutics)
  • a single NDC of the present disclosure may include up to about 80 drug molecules on each nanoparticle (e.g., 80 exatecan molecules).
  • conventional ADCs only about 4 to 8 therapeutic/drug molecules can be attached to the antibody, and conventional FR-targeted small molecule drugs are limited to only a single therapeutic/drug molecule.
  • the NDCs described herein can carry at least 10 times more drug molecules NDC, relative to conventional drug delivery platforms, and deliver a relatively higher drug load to cells.
  • FR-targeting drug-delivery platforms such as ADCs and FR-targeted small molecular chemotherapeutics
  • ADCs and FR-targeted small molecular chemotherapeutics usually exhibit high potency in cancer cells with high receptor expression level, their efficacy in cancer cells with medium or low FR expression level is limited.
  • the NDCs of the present disclosure can effectively target cancer cells with both high and low FR expression levels and provide potent therapy for cancers that have low FR expression ( see, e.g., FIG. 23 and associated assay described in the Examples).
  • the NDCs disclosed herein can include multiple FR-targeting ligands on a single nanoparticle, there is a multivalent or avidity effect on binding to several FRs on the cell surface.
  • a single ADC generally can only bind to up to two FRs on the cell surface, and a single FR-targeted chemotherapy drug can only bind to one FR on the cell surface.
  • the multivalent effect of the FR-targeted NDCs of the present disclosure can significantly enhance the binding of NDC to cells that express FR, leading to improved targeting efficiency and therapeutic outcomes.
  • This multivalent effect can also render the NDCs of the present disclosure suitable for treating cancers that have low FR-expression, that cannot be effectively treated using conventional FR-targeted drug delivery platforms, such as ADCs or FR-targeted chemotherapy drugs.
  • ADCs in solid tumor treatment are usually greatly limited by their poor tumor penetration.
  • the FR-targeted NDCs disclosed herein exhibit highly effective tumor penetration, permitting the delivery of therapeutics throughout a tumor following administration, which improves therapeutic outcomes in treating solid tumors, relative to the use of ADCs.
  • the NDCs of the present disclosure have a smaller size than conventional drug delivery platforms, such as ADCs.
  • the NDCs of the present disclosure are smaller than the particle size cut off for renal clearance, permitting the NDC to be renally clearable.
  • NDCs that are administered to a subject but do not enter a cancer cell i.e., non-targeted NDCs
  • This target- and/or-clear approach reduces the toxicity of NDCs as compared to conventional drug delivery platforms, such as ADCs, and prevents undesirable accumulation of the NDCs (or their payloads) in healthy tissues or organs.
  • the NDCs of the present disclosure exhibit improved biodistribution than conventional drug delivery platforms, such as ADCs, resulting in reduced side effect and toxicity.
  • NDCs comprising a nanoparticle, such as a silica nanoparticle.
  • the nanoparticle may comprise a silica-based core and a silica shell surrounding at least a portion of the core.
  • the nanoparticle may have only the core and no shell.
  • the core of the nanoparticle may contain the reaction product of a reactive fluorescent compound and a co-reactive organo-silane compound.
  • the core of the nanoparticle may contain the reaction product of a reactive fluorescent compound and a co-reactive organo-silane compound, and silica.
  • the nanoparticle is a core-shell particle.
  • the diameter of the core may be from about 0.5 nm to about 100 nm, from about 0.1 nm to about 50 nm, from about 0.5 nm to about 25 nm, from about 0.8 nm to about 15 nm, or from about 1 nm to about 8 nm.
  • the diameter of the core may be from about 3 nm to about 8 nm, or 3 nm to about 6 nm, e.g., the diameter of the core may be from about 3 nm to about 4 nm, about 4 nm to about 5 nm, about 5 nm to about 6 nm, about 6 nm to about 7 nm, or about 7 nm to about 8 nm.
  • the shell of the nanoparticle can be the reaction product of a silica forming compound, such as a tetraalkyl orthosilicate, for example tetraethyl orthosilicate (TEOS).
  • a silica forming compound such as a tetraalkyl orthosilicate, for example tetraethyl orthosilicate (TEOS).
  • TEOS tetraethyl orthosilicate
  • the shell of the nanoparticle may have a range of layers.
  • the silica shell may be from about 1 to about 20 layers, from about 1 to about 15 layers, from about 1 to about 10 layers, or from about 1 to about 5 layers.
  • the silica shell may comprise from about 1 to about 3 layers.
  • the thickness of the shell may range from about 0.5 nm to about 90 nm, from about 0.5 nm to about 40 nm, from about 0.5 nm to about 20 nm, from about 0.5 nm to about 10 nm, or from about 0.5 nm to about 5 nm, e.g., about 1 nm, about 2 nm, about 3 nm, about 4 nm, or about 5 nm.
  • the thickness of the silica shell may be from about 0.5 nm to about 2 nm.
  • the silica shell of the nanoparticle may cover only a portion of nanoparticle or the entire particle.
  • the silica shell may cover about 1 to about 100 percent, from about 10 to about 80 percent, from about 20 to about 60 percent, or from about 30 to about 50 percent of the nanoparticle.
  • the silica shell may cover about 50 to about 100 percent.
  • the silica shell can be either solid, i.e., substantially non-porous, meso-porous, semi-porous, or the silica shell may be porous.
  • the silica nanoparticle can be either solid, i.e., substantially non-porous, meso-porous, semi-porous, or the silica nanoparticle may be porous.
  • the nanoparticle is a non-mesoporous nanoparticle, e.g., a non-mesoporous silica nanoparticle, such as a non-mesoporous silica core-shell nanoparticle.
  • the surface of the nanoparticle may be modified to incorporate at least one functional group.
  • An organic polymer may be attached to the nanoparticle and can be modified to incorporate at least one functional group by any known techniques in the art.
  • the functional groups can include, but are not limited to, dibenzocyclooctyne (DBCO), maleimide, N-hydroxysuccinimide (NHS) ester, a diene (e.g., cyclopentadiene), an amine, or a thiol.
  • a bifunctional group comprising a silane at one terminus, and a DBCO, maleimide, NHS ester, diene (e.g., cyclopentadiene), amine, or thiol at the other terminus, may be condensed onto the surface of the silica nanoparticle via the silane group.
  • the incorporation of the functional group can also be accomplished through known techniques in the art, such as using "click chemistry," amide coupling reactions, 1,2-additions such as a Michael addition, or Diels-Alder (2+4) cycloaddition reactions. This incorporation allows attachment of various targeting ligands, contrast agents and/or therapeutic agents to the nanoparticle.
  • the organic polymers that may be attached to the nanoparticle include, but are not limited to, polyethylene glycol) (PEG), polylactate, polylactic acids, sugars, lipids, polyglutamic acid (PGA), polyglycolic acid, poly(lactic-co-glycolic acid) (PLGA), polyvinyl acetate (PVA), and combinations thereof.
  • the organic polymer is poly(ethylene glycol) (PEG).
  • the surface of the nanoparticle is functionalized.
  • the surface of the nanoparticle can have functional groups other than those resulting from the synthesis of the nanoparticles (e.g., -OH groups (resulting from terminal Si-OH groups on a nanoparticle surface) and PEG groups (resulting from Si-PEG groups on the nanoparticle surface).
  • -OH groups resulting from terminal Si-OH groups on a nanoparticle surface
  • PEG groups resulting from Si-PEG groups on the nanoparticle surface
  • the nanoparticle may comprise a non-pore surface and a pore surface.
  • at least a portion of the individual nanoparticle non-pore surface and at least a portion of the individual nanoparticle pore surface are functionalized.
  • at least a portion of the nanoparticle non-pore surface and the at least a portion of the pore surface have different functionalization.
  • the pore surface is also referred to herein as the interior surface.
  • the nanoparticles may also have a non-pore surface (or non-porous surface).
  • the non-pore surface is also referred to herein as the exterior nanoparticle surface.
  • the pore surface (e.g., at least a portion of the pore surface) and/or the non-pore surface (e.g., at least a portion of the non-pore surface) of the nanoparticle can be functionalized.
  • the nanoparticles can be reacted with compounds such that a functional group of the compound is presented on (e.g., covalently bonded to) the surface of the nanoparticle.
  • the surface can be functionalized with hydrophilic groups (e.g., polar groups such as ketone groups, carboxylic acid, carboxylate groups, and ester groups), which provide a surface having hydrophilic character, or hydrophobic groups (e.g., nonpolar groups such as alkyl, aryl, and alkylaryl groups), which provide a surface having hydrophobic character.
  • hydrophilic groups e.g., polar groups such as ketone groups, carboxylic acid, carboxylate groups, and ester groups
  • hydrophobic groups e.g., nonpolar groups such as alkyl, aryl, and alkylaryl groups
  • DEDMS diethoxydimethylsilane
  • the surface of the nanoparticle is at least partially functionalized with polyethylene glycol (PEG) groups.
  • PEG polyethylene glycol
  • the attachment of PEG to the nanoparticle may be accomplished by a covalent bond or a non-covalent bond, such as by ionic bond, hydrogen bond, hydrophobic bond, coordination, adhesive, and physical absorption.
  • the PEG groups are attached (e.g., covalently attached) to the surface of the nanoparticle.
  • the PEG groups are covalently bonded to the silica at the surface of the shell via a Si-O-C bond and or to the silica in the core.
  • the PEG groups are covalently bonded to the silica in the core.
  • the nanoparticle is a core-shell nanoparticle, wherein the PEG groups are covalently bonded to the silica at the surface of the shell via a Si-O-C bond.
  • the PEG groups on the nanoparticle surface can prevent adsorption of serum proteins to the nanoparticle in a physiological environment (e.g., in a subject), and may facilitate efficient urinary excretion and decrease aggregation of the nanoparticle ( see, e.g., Bums et al. "Fluorescent silica nanoparticles with efficient urinary excretion for nanomedicine", Nano Letters (2009) 9(1):442-448 ).
  • the PEG groups may be derived from PEG polymer having a molecular weight (Mw) of 400 g/mol to 2000 g/mol, including all integer g/mol values and ranges therebetween.
  • the PEG groups are derived from PEG polymer having a Mw of 460 g/mol to 590 g/mol, which contain 6 to 9 ethylene glycol units.
  • the nanoparticles are at least 50%, at least 75%, at least 90%, or at least 95% functionalized with PEG groups.
  • the nanoparticles are functionalized with PEG groups with the maximum number of PEG groups such that, the pores remain accessible (e.g., the pores can be functionalized).
  • the pore surface is a silica surface having terminal silanol (Si-OH) groups.
  • a polyethylene glycol unit disclosed herein may be functionalized with a functional group, for example, a "click chemistry" group, such as dibenzocyclooctyne (DBCO) or azide, a diene (e.g., cyclopentadiene), a maleimide, an NHS ester, an amine, a thiol, or an activated acetylene moiety such as While DBCO can be used, the functional group may also be another alkyne, such as another strained alkyne (e.g., DIBO or a derivative thereof, or a derivative of DBCO). Also, the functional group may be a nitrone or a nitrile oxide.
  • a “click chemistry” group such as dibenzocyclooctyne (DBCO) or azide
  • a diene e.g., cyclopentadiene
  • a maleimide e.g., an NHS ester, an amine, a
  • an NDC may be functionalized with a functional group such as a "click chemistry" group, e.g., dibenzocyclooctyne (DBCO) or azide; a diene (e.g., cyclopentadiene); a maleimide; an NHS ester; an amine; a thiol; or an activated acetylene moiety such as that may comprise any suitable linker, or may have no linker.
  • DBCO dibenzocyclooctyne
  • the functional group may also be another alkyne, such as another strained alkyne (e.g., DIBO or a derivative thereof, or a derivative of DBCO). Also, the functional group may be a nitrone or a nitrile oxide.
  • another alkyne such as another strained alkyne (e.g., DIBO or a derivative thereof, or a derivative of DBCO).
  • the functional group may be a nitrone or a nitrile oxide.
  • a DBCO-functionalized linker may be introduced to a nanoparticle (e.g., a PEGylated C'Dot) by reacting the silane group on a DBCO-linker-silane compound with a silanol group on the surface of the nanoparticle (e.g., under the PEG layer on the C'Dot surface).
  • a nanoparticle e.g., a PEGylated C'Dot
  • a diene-functionalized precursor e.g., cyclopentadiene-functionalized precursor
  • a nanoparticle e.g., a PEGylated C'Dot
  • reacting the silane group on a diene-linker-silane or diene-silane precursor compound with a silanol group on the surface of the nanoparticle e.g., under the PEG layer on the C'Dot surface
  • a second precursor that comprises a reactive group (e.g., DBCO) via a dienophile.
  • DBCO reactive group
  • the linker group in the DBCO-linker-silane or diene-linker-silane can comprise any structure (or sub-structure), including but not limited to PEG, a carbon chain (e.g., alkylene), a heteroalkylene group, or the like.
  • the diene-functionalized linker covalently attached to the nanoparticle may be further modified, e.g., by reaction with a DBCO-functionalized group.
  • the diene-functionalized linker covalently attached to the nanoparticle may be contacted with a DBCO-linker-maleimide compound (or other suitable DBCO-linker-dienophile), to form a cycloadduct between the diene and maleimide, resulting in an NDC comprising DBCO groups attached to its surface, e.g., using cycloaddition chemistry, such as a Diels-Alder cycloaddition.
  • a DBCO-linker-maleimide compound or other suitable DBCO-linker-dienophile
  • Functionalization facilitates the conjugation of suitably functionalized FR-targeting ligands and/or functionalized drug payloads (such as azide-functionalized FR-targeting ligands and/or azide-functionalized drug payloads) to the nanoparticle by a coupling reaction, e.g., via click chemistry, (3+2) cycloaddition reactions, amide coupling, or Diels-Alder reaction.
  • a coupling reaction e.g., via click chemistry, (3+2) cycloaddition reactions, amide coupling, or Diels-Alder reaction.
  • NDCs disclosed herein can be prepared using relatively stable linker or spacer groups, or precursors thereof.
  • the linker or spacer groups, or their precursors can avoid premature or undesired cleavage, which can occur using other linkers or precursors.
  • certain methods of functionalizing nanoparticles employ amine-silane precursors (to provide amine-functionalized nanoparticles) that are modified at the amine groups to conjugate other moieties to the nanoparticle.
  • the amine-silane precursors can be unstable and can self-condense during reaction, causing undesired aggregation. The aggregates can be very difficult to separate from the functionalized nanoparticles.
  • the amine groups on the surface of the nanoparticle can promote undesired reactivity, that may lead to premature release of the payload, or undesired release of the targeting ligand.
  • the NDCs disclosed herein can be produced using relatively stable precursors, and the NDCs are stable and highly pure.
  • the nanoparticles of the present NDCs can be prepared with a silane-diene precursor (such as a silane-cyclopentadiene precursor), to afford a nanoparticle functionalized with one or more diene groups.
  • the diene groups may then be reacted with a second precursor, such as a dienophile-containing precursor (e.g., a PEG-maleimide derivative, e.g., a DBCO-PEG-maleimide), causing a stable cycloadduct to form.
  • a dienophile-containing precursor e.g., a PEG-maleimide derivative, e.g., a DBCO-PEG-maleimide
  • the resulting functionalized nanoparticle comprising the cycloadduct, may optionally be reacted with one or more subsequent precursors (such as targeting ligand precursors and/or payload-linker conjugate precursors described herein), to further functionalize the nanoparticle.
  • the diene-silane precursors, and the cycloadducts that are produced do not exhibit the undesired qualities of other functionalized nanoparticles, e.g., they have relatively high serum stability, can be produced in high yield and purity (e.g., free of aggregated precursor). See, e.g., FIGS. 33A-33B .
  • any desired ratio of payload, targeting ligand, or otherwise can be introduced to the nanoparticle. Examples of preparing nanoparticles using these methods, and their benefits, are provided in the Examples.
  • the NDCs of the present disclosure may comprise a structure of Formula (NP): wherein x is an integer of 0 to 20, e.g., 4; wherein the silicon atom is a part of the nanoparticle; and wherein the adjacent to the triazole moiety denotes a point of attachment to a targeting ligand or payload-linker conjugate, either directly or indirectly, e.g., via a linker or spacer group, e.g., a PEG moiety.
  • NP Formula
  • the attachment may be to a linker or spacer group, e.g., the linker of a linker-payload conjugate, or a linker or spacer group of a folate receptor targeting ligand, e.g., a PEG moiety.
  • the NDCs of the present disclosure may be prepared from diene (e.g., cyclopentadiene) functionalized nanoparticles, e.g., by conjugating a linker moiety (e.g., a linker comprising a dienophile, such as maleimide) to the diene with a cycloaddition reaction.
  • the silica shell surface of the nanoparticles can be modified by using known cross-linking agents to introduce surface functional groups.
  • Crosslinking agents include, but are not limited to, divinyl benzene, ethylene glycol dimethacrylate, trimethylol propane trimethacrylate, N,N'-methylene-bis-acrylamide, alkyl ethers, sugars, peptides, DNA fragments, or other known functionally equivalent agents.
  • the nanoparticle may also be conjugated to a contrast agent, such as a radionuclide.
  • a contrast agent such as a radionuclide
  • the nanoparticles may incorporate any suitable fluorescent compound, such as a fluorescent organic compound, a dye, a pigment, or a combination thereof.
  • fluorescent compounds can be incorporated into the silica matrix of the core of the nanoparticle.
  • suitable chemically reactive fluorescent dyes/fluorophores are known, see for example, MOLECULAR PROBES HANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS, 6th ed., R. P. Haugland, ed. (1996 ).
  • the fluorescent compound is covalently encapsulated within the core of the nanoparticle.
  • fluorescent compound can be, but is not limited to, a near infrared fluorescent (NIRF) dye that is positioned within the silica core of the nanoparticle, that can provide greater brightness and fluorescent quantum yield relative to the free fluorescent dye.
  • NIRF near infrared fluorescent
  • the near infrared-emitting probes exhibit decreased tissue attenuation and autofluorescence ( Burns et al. "Fluorescent silica nanoparticles with efficient urinary excretion for nanomedicine", Nano Letters (2009) 9(1):442-448 ).
  • Fluorescent compounds that may be used (e.g., encapsulated by an NDC) in the present disclosure, include, but are not limited to, Cy5, Cy5.5 (also known as Cy5++), Cy2, fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin, Cy7, fluorescein (FAM), Cy3, Cy3.5 (also known as Cy3++), Texas Red (sulforhodamine 101 acid chloride), LIGHTCYCLER ® -Red 640, LIGHTCYCLER ® -Red 705, tetramethylrhodamine (TMR), rhodamine, rhodamine derivative (ROX), hexachlorofluorescein (HEX), rhodamine 6G (R6G), the rhodamine derivative JA133, Alexa Fluorescent Dyes (such as ALEXA FLUOR ® 488, ALEXA
  • Fluorescent compounds that can be used also include fluorescent proteins, such as GFP (green fluorescent protein), enhanced GFP (EGFP), blue fluorescent protein and derivatives (BFP, EBFP, EBFP2, azurite, mKalama1), cyan fluorescent protein and derivatives (CFP, ECFP, Cerulean, CyPet) and yellow fluorescent protein and derivatives (YFP, Citrine, Venus, YPet) ( WO 2008/142571 , WO 2009/056282 , WO 1999/22026 ).
  • fluorescent proteins such as GFP (green fluorescent protein), enhanced GFP (EGFP), blue fluorescent protein and derivatives (BFP, EBFP, EBFP2, azurite, mKalama1)
  • CFP cyan fluorescent protein and derivatives
  • CFP ECFP, Cerulean, CyPet
  • YFP Citrine, Venus, YPet
  • the fluorescent compound is selected from the group consisting of Cy5 and Cy5.5. In preferred aspects, the fluorescent compound is Cy5.
  • a fluorescent nanoparticle may be synthesized by the steps of: (1) covalently conjugating a fluorescent compound, such as a reactive fluorescent dye (e.g., Cy5), with a reactive moiety including, but not limited to, maleimide, iodoacetamide, thiosulfate, amine, N-hydroxysuccimide ester, 4-sulfo-2,3,5,6-tetrafluorophenyl (STP) ester, sulfosuccinimidyl ester, sulfodichlorophenol esters, sulfonyl chloride, hydroxyl, isothiocyanate, carboxyl, to an organo-silane compound, such as a co-reactive organo-silane compound, to form a fluorescent silica precursor, and reacting the fluorescent silica precursor to form a fluorescent core; or (2) reacting the fluorescent silica precursor with a silica forming compound, such as tetraalkoxysilane,
  • Fluorescent silica-based nanoparticles are known in the art and are described by US 8298677 B2 , US 9625456 B2 , US 10548997 B2 , US 9999694 B2 , US 10039847 B2 and US 10548998 B2 , the contents of which are each incorporated herein by reference in their entireties.
  • the NDCs comprise a nanoparticle that comprises a silica-based core and a silica shell surrounding at least a portion of the core and polyethylene glycol (PEG) is covalently bonded to the surface of the nanoparticle, and a fluorescent compound is covalently encapsulated within the core of the nanoparticle.
  • PEG polyethylene glycol
  • the NDCs of the present disclosure may comprise a targeting ligand that is attached to the nanoparticle directly or indirectly through a spacer group.
  • NDCs with targeting ligands can enhance internalization of the payload/drugs in tumor cells and/or deliver drugs into tumor cells due to increased permeability, as well as the targeting ability of the NDC.
  • the targeting ligand can allow the nanoparticle to target a specific cell type through the specific binding between the ligand and the cellular component.
  • the targeting ligand may also facilitate entry of the nanoparticle into the cell or barrier transport, for example, for assaying the intracellular environment.
  • the targeting ligands of the present disclosure are capable of binding to receptors on tumor cells.
  • the targeting ligands can bind to the folate receptor (FR), including all four human isoforms of FR, including FR alpha (FR ⁇ , also known as FOLR1), FR beta (FR ⁇ , also known as FOLR2), FR gamma (FR ⁇ , also known as FOLR3), and FR delta (FR ⁇ , also known as FOLR4).
  • FR alpha FR ⁇ , also known as FOLR1
  • FR ⁇ also known as FOLR2
  • FOLR3 FR gamma
  • FOLR4 FR delta
  • Conjugation of FR targeting ligand to the surface of the nanoparticle of the present disclosure allows for targeted therapy of FR-overexpressing cancerous cells, tissues, and tumors.
  • NDCs of the present disclosure comprising targeting ligands that can bind to folate receptor alpha (FR ⁇ ), such as folic acid, may be used for targeting ovarian cancer, endometrial cancer, fallopian tube cancer, peritoneal cancer, cervical cancer, breast cancer, lung cancer, mesothelioma, uterine cancer, gastrointestinal cancer (e.g., esophageal cancer, colon cancer, rectal cancer, and stomach cancer), pancreatic cancer, bladder cancer, kidney cancer, liver cancer, head and neck cancer, brain cancer, thyroid cancer, skin cancer, prostate cancer, and testicular cancer, acute myeloid leukemia (AML, e.g., pediatric AML).
  • AML acute myeloid leukemia
  • NDCs of the present disclosure comprising targeting ligands that can bind to folate receptor beta (FR ⁇ ) may be used for targeting acute myeloid leukemia (AML, e.g., pediatric AML), chronic myelogenous leukemia (CML), and tumor associated macrophages.
  • AML acute myeloid leukemia
  • CML chronic myelogenous leukemia
  • Tumor associated macrophages can be targeted as a means to modify the immune status of the tumor.
  • the binding affinity of FR-targeted NDCs to folate receptors can be enhanced due to multivalence effect.
  • Folate receptor can be highly expressed in solid tumor cells, including ovarian, kidney, lung, brain, endometrial, colorectal, pancreatic, gastric, prostate, breast and non-small-cell lung cancers.
  • FR is over-expressed in other cancers including fallopian tube cancer, cervical cancer, mesothelioma, uterine cancer, esophageal cancer, stomach cancer, bladder cancer, liver cancer, head and neck cancer, thyroid cancer, skin cancer, and testicular cancer.
  • FR is also over-expressed in hematological malignancies, such as acute myeloid leukemia (AML) and chronic myelogenous leukemia (CML).
  • AML acute myeloid leukemia
  • CML chronic myelogenous leukemia
  • the targeting ligands bind to folate receptor alpha (FR ⁇ ), folate receptor beta (FR ⁇ ), or both.
  • FR-targeting ligands that are capable of binding to specific cell types having elevated levels of FR ⁇ , such as, but not limited to, cancer (e.g., adenocarcinomas) of uterus, ovary, breast, cervix, kidney, colon, testicles (e.g., testicular choriocarcinoma), brain (e.g., ependymal brain tumors), malignant pleural mesothelioma, and nonfunctioning pituitary adenocarcinoma.
  • cancer e.g., adenocarcinomas
  • ovary ovary
  • breast cervix
  • kidney colon
  • testicles e.g., testicular choriocarcinoma
  • brain e.g., ependymal brain tumors
  • malignant pleural mesothelioma e.g., ependymal brain tumors
  • the present disclosure also provides FR-targeting ligands that are capable of targeting acute myeloid leukemia (AML, e.g., pediatric AML), chronic myelogenous leukemia (CML), and tumor associated macrophages.
  • the targeting ligand can be any suitable molecule that can bind a FR, such as FR ⁇ , such as a small organic molecule (e.g., folate or a folate analog), an antigen-binding portion of an antibody (e.g.
  • Fab fragment a Fab' fragment, a F(ab')2 fragment, a scFv fragment, a Fv fragment, a dsFv diabody, a dAb fragment, a Fd' fragment, a Fd fragment, or an isolated complementarity determining region (CDR) region
  • an antibody mimetic e.g., aptamer, an affibody, affilin, affimer, anticalin, avimer, Darpin, and the like
  • nucleic acid lipid, and the like.
  • the targeting ligand is folic acid, or a folate receptor binding derivative thereof.
  • folic acid can encompass any amide or ester derivative of folic acid.
  • free folic acid may be modified to be conjugated to the nanoparticle via a spacer group, such as PEG or a PEG derivative (e.g., by forming an amide bond between the terminal carboxylic acid of folic acid, and a nitrogen atom of the spacer group).
  • the FR-targeted NDCs may not only accumulate in a cancer cell or tumor, but may also penetrate the tumor tissue and deliver payloads to the entire tumor tissue for optimal treatment efficacy. Without wishing to be bound by any particular theory or mechanism, it is believed that the targeting ligands bind to the specific receptor groups on the surface of the cancer cell, resulting in receptor-mediated cell uptake of NDCs. This receptor-mediated cell uptake of NDCs happens via the endocytosis process, and eventually traffics NDCs to endosomes and lysosomes in cancer cells.
  • the NDCs comprise a targeting ligand that is attached to the nanoparticle directly or indirectly through a spacer group.
  • the targeting ligand can be attached to the nanoparticle directly via the silica of the nanoparticle (i.e., covalently bonded).
  • the targeting ligand is attached to the nanoparticle indirectly through a suitable spacer group.
  • the spacer group can be any group that can act as a spacer, e.g., as a spacer between a targeting ligand and the nanoparticle, and attach the targeting ligand to the nanoparticle.
  • the spacer group may be a divalent linker, such as a divalent linker that comprises a chain length of between about 5 and about 200 atoms (e.g., carbon atoms, heteroatoms, or a combination thereof), such as between about 5 and about 100 atoms, between about 5 and about 80 atoms, between about 10 and about 80 atoms, between about 10 and about 70 atoms, between about 10 and about 30 atoms, between about 20 and about 30 atoms, between about 30 and about 80 atoms, or between about 30 and about 60 atoms.
  • a divalent linker such as a divalent linker that comprises a chain length of between about 5 and about 200 atoms (e.g., carbon atoms, heteroatoms, or a combination thereof), such as between about
  • Suitable spacer groups may comprise an alkylene, alkenylene, alkynylene, heteroalkylene (e.g., PEG), carbocyclyl, heterocyclyl, aryl, heteroaryl, or a combination thereof.
  • the spacer group may comprise a PEG group, an alkylene group, or a combination thereof.
  • the spacer group may be substituted or unsubstituted, e.g., the spacer group may comprise a substituted alkylene, substituted heteroalkylene, or a combination thereof.
  • the spacer group may comprise a PEG group (or PEG spacer), an alkylene group (or alkylene spacer), one or more heteroatoms, and/or one or more cyclic groups (e.g., heterocyclylene groups, such as a piperazine).
  • the targeting ligand such as folic acid
  • the folic acid may be present in the NDC as an amide, e.g., to facilitate conjugation to a PEG spacer group or other divalent linker, e.g., as shown in FIG. 1 .
  • the number of PEG monomers in a PEG spacer may range from 2 to 20, from 2 to 10, from 2 to 8, or from 2 to 5.
  • the number of PEG groups as spacers in a functionalized FR-targeting ligand is 3.
  • the average nanoparticle to targeting ligand (e.g., folic acid) ratio may range from about 1 to about 50, from about 1 to about 40, from about 1 to about 30, or from about 1 to about 20.
  • the average nanoparticle to targeting ligand (e.g., folic acid) ratio may be about 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, 1:30, 1:40, or 1:50.
  • the average nanoparticle to targeting ligand ratio may range from about 1 to about 20, e.g., the average number of folic acid molecules on each nanoparticle may be between about 5 and about 10, between about 10 and about 15, or between about 15 and about 20, e.g., about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, or about 15 folic acid molecules per nanoparticle.
  • An NDC disclosed herein may comprise about 10 folic acid molecules.
  • An NDC disclosed herein may comprise about 11 folic acid molecules.
  • An NDC disclosed herein may comprise about 12 folic acid molecules.
  • An NDC disclosed herein may comprise about 13 folic acid molecules.
  • An NDC disclosed herein may comprise about 14 folic acid molecules.
  • An NDC disclosed herein may comprise about 15 folic acid molecules.
  • a smaller number of targeting ligands attached to the nanoparticle may help maintain the hydrodynamic diameter of the nanoparticle, e.g., to meet the renal clearance cutoff size range ( Hilderbrand et al., Near-infrared fluorescence: Application to in vivo molecular imaging, Curr. Opin. Chem. Biol., (2010) 14:71-79 ).
  • the number of targeting ligands measured may be an average number of targeting ligands attached to more than one nanoparticle. Alternatively, one nanoparticle may be measured to determine the number of targeting ligands attached.
  • the number of targeting ligands attached to the nanoparticle can be measured by any suitable methods, such as, but not limited to, optical imaging, fluorescence correlation spectroscopy (FCS), UV-Vis, chromatography, mass spectroscopy, or indirect enzymatic analysis.
  • FCS fluorescence correlation spectroscopy
  • UV-Vis UV-Vis
  • chromatography chromatography
  • mass spectroscopy or indirect enzymatic analysis.
  • the targeting ligand can be attached to the nanoparticle via covalent bonding to the silica of the nanoparticle (e.g., indirectly through a spacer group).
  • the ligand may be conjugated to a nanoparticle (e.g., via a functional group on the nanoparticle surface) described herein, for example, using coupling reactions, Click Chemistry (e.g., a 3+2 Click Chemistry reaction), cycloaddition (e.g., a 3+2 or 2+4 cycloaddition reaction, using the appropriate functional groups), or conjugation via a carboxylate, ester, alcohol, carbamide, aldehyde, amine, sulfur oxide, nitrile oxide, nitrone, nitrogen oxide, halide, or any other suitable compound known in the art.
  • Click Chemistry e.g., a 3+2 Click Chemistry reaction
  • cycloaddition e.g., a 3+2 or 2+4 cycloaddition reaction, using the appropriate functional groups
  • the conjugation of FR-targeting ligands can be accomplished by "click chemistry” reaction using a diarylcyclooctyne (DBCO) group.
  • DBCO diarylcyclooctyne
  • Any suitable reaction mechanism may be adapted in the present disclosure for "click chemistry", so long as facile and controlled attachment of the targeting ligand to the nanoparticle can be achieved.
  • a triple bond (e.g., alkyne, e.g., terminal alkyne) is introduced onto the surface of a nanoparticle (e.g., via a PEG covalently conjugated with the shell of the nanoparticle, or through another suitable linker or spacer group).
  • an azide bond, or other group that is reactive with a triple bond may be introduced onto the desired targeting ligand.
  • folic acid may be modified by conjugating the terminal carboxylic acid of folic acid with a spacer group (e.g., a PEG moiety), that comprises an azide at one terminus).
  • the nanoparticle e.g., PEGylated nanoparticle
  • the targeting ligand comprising a group reactive with the triple bond
  • the targeting ligand e.g., "Click Chemistry”
  • An azide functionalized FR-ligand (where the FR-ligand may comprise a spacer group, and the spacer group may possess the azide group) can be attached to the nanoparticle either directly or indirectly via an alkyne (e.g., DBCO group).
  • Spacer groups such as, but not limited to PEG groups, can be present in a FR-targeting ligand precursor, and may possess a terminal group (e.g., azide) to facilitate conjugation to the nanoparticle, and after conjugation, the spacer group may be disposed between the targeting ligand and the nanoparticle.
  • the FR-targeting ligand precursor may comprise a structure of Formula (D-1): wherein y is an integer of 0 to 20 (e.g., 3).
  • y may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, e.g., 2, 3, or 4.
  • the FR-targeting ligand may be functionalized with a suitable terminal group, such as, but not limited to an azide group.
  • the azide functionalized FR-ligand can be attached to the nanoparticle either directly or indirectly via the DBCO groups.
  • Spacer groups such as, but not limited to PEG groups can be present between the azide functionalized FR-ligand and the nanoparticle.
  • the FR-targeting ligand is functionalized to include spacer groups, such as, but not limited to PEG groups that terminate with an azide group that reacts with the DBCO groups on the surface of the nanoparticle.
  • FR-targeting ligand may include hydrophilic PEG groups as spacers, that may enhance solubility in water, and may reduce or eliminate aggregation and precipitation of the nanoparticle.
  • the number of PEG groups as spacers that can be present in a functionalized FR-targeting ligand may be in the range of from 2 to 20, from 2 to 10, from 2 to 8, or from 2 to 5. In preferred aspects, the number of PEG groups as spacers in a functionalized FR-targeting ligand is 3.
  • the NDCs of the present disclosure comprising a targeting ligand may comprise a structure of Formula (NP-2): wherein x is an integer of 0 to 10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, e.g., 4), and y is an integer of 0 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, e.g., 3), and the silicon atom is a part of the nanoparticle (e.g., bonded with the silica shell of a core-shell silica nanoparticle).
  • x may be 4, and y may be 3.
  • Each nanoparticle of the NDCs disclosed herein may comprise more than one molecule of Formula (NP-2), for example, the nanoparticle may comprise between about 1 and about 20 molecules of Formula (NP-2), e.g., between about 5 and about 20 molecules of Formula (NP-2), between about 8 and about 15 molecules of Formula (NP-2), between about 10 and about 15 molecules of Formula (NP-2), e.g., about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 molecules of Formula (NP-2).
  • An NDC disclosed herein may comprise about 12 molecules of Formula (NP-2).
  • An NDC disclosed herein may comprise about 13 molecules of Formula (NP-2).
  • the NDCs of the present disclosure can also comprise a linker-payload conjugate that is attached to the nanoparticle directly or indirectly through a spacer group.
  • the linker-payload conjugate is attached to the nanoparticle through a spacer group.
  • the payload may be exatecan, or a salt or analog thereof.
  • the spacer group can be any group that can act as a spacer, e.g., as a spacer between a payload/linker conjugate and the nanoparticle, and attach the linker-payload conjugate to the nanoparticle.
  • the spacer group may be a divalent linker, such as a divalent linker that comprises a chain length of between about 5 and about 200 atoms (e.g., carbon atoms, heteroatoms, or a combination thereof), such as between about 5 and about 100 atoms, between about 5 and about 80 atoms, between about 10 and about 80 atoms, between about 10 and about 70 atoms, between about 10 and about 30 atoms, between about 20 and about 30 atoms, between about 30 and about 80 atoms, or between about 30 and about 60 atoms.
  • a divalent linker such as a divalent linker that comprises a chain length of between about 5 and about 200 atoms (e.g., carbon atoms, heteroatoms, or a combination
  • Suitable spacer groups may comprise an alkylene, alkenylene, alkynylene, heteroalkylene (e.g., PEG), carbocyclyl, heterocyclyl, aryl, heteroaryl, or a combination thereof.
  • the spacer group may comprise a PEG group, an alkylene group, or a combination thereof.
  • the spacer group may be substituted or unsubstituted, e.g., the spacer group may comprise a substituted alkylene, substituted heteroalkylene, or a combination thereof.
  • the spacer group may comprise a PEG group (or PEG spacer), an alkylene group (or alkylene spacer), one or more heteroatoms, and/or one or more or cyclic groups.
  • a functional group e.g., amine, hydroxyl, or sulfhydryl
  • the payload e.g., exatecan
  • an existing functional group on the payload e.g., pendant amine group
  • exatecan contains an amine functional group suitable for coupling to the linker moiety
  • the payload (e.g., exatecan payload, or a salt or analog thereof) can be cleaved from the nanoparticle inside a cell, or a cell organelle, e.g., by an enzyme, thereby releasing exatecan, e.g., inside the cell or cell organelle).
  • Exatecan is a topoisomerase 1 (Topo-1) inhibitor that can stabilize the complexes of DNA and Topo-1 enzyme, preventing DNA relegation and inducing lethal DNA strand breaks. The generation of these DNA lesions is effective for killing cancer cells, allowing NDCs of the present disclosure to achieve the desired therapeutic effect.
  • Topo-1 topoisomerase 1
  • the payload is exatecan, or a salt thereof. In other preferred aspects of the present disclosure, the payload is an analog of exatecan, or a salt thereof.
  • the average nanoparticle to payload ratio ranges from 1 to 80, from 1 to 70, from 1 to 60, from 1 to 50, from 1 to 40, from 1 to 30, from 1 to 20, from 1 to 15, from 1 to 12 and preferably from 1 to 10.
  • the average nanoparticle to payload (e.g., exatecan, or a salt or analog thereof) ratio may be about 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, 1:30, 1:32, 1:34, 1:36, 1:38, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, or 1:80.
  • the average nanoparticle to payload e.g., exatecan, or a salt or analog thereof
  • the average number of exatecan molecules on each nanoparticle may be between about 5 and about 10, between about 10 and about 15, between about 15 and about 20, between about 20 and about 25, or between about 25 and about 30, e.g., about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, or about 30 exatecan molecules per nanoparticle.
  • An NDC disclosed herein may comprise about 18 exatecan molecules.
  • An NDC disclosed herein may comprise about 19 exatecan molecules.
  • An NDC disclosed herein may comprise about 20 exatecan molecules.
  • An NDC disclosed herein may comprise about 21 exatecan molecules.
  • An NDC disclosed herein may comprise about 22 exatecan molecules.
  • An NDC disclosed herein may comprise about 23 exatecan molecules.
  • An NDC disclosed herein may comprise about 24 exatecan molecules.
  • An NDC disclosed herein may comprise about 25 exatecan molecules.
  • An NDC disclosed herein may comprise about 26 exatecan molecules.
  • An NDC disclosed herein may comprise about 27 exatecan molecules.
  • Vintafolide developed by Endocyte and Merck & Co. is a small molecule drug conjugate consisting of a small molecule targeting the Folate Receptor, which is over expressed on certain cancers, such as ovarian cancer, and a chemotherapy drug, Vinblastine ( US 7601332 B2 and US 1002942 B2 ).
  • vintafolide is capable of carrying single molecule of payload only, attached to the targeting moiety by a pH-cleavable linker.
  • several cytotoxic payloads e.g., exatecan molecules
  • the linkers in the linker-payload conjugates can be self-immolative linkers that are capable of releasing the active payload in vitro as well as in vivo under conditions sufficient for enzymatic release of the active payload (e.g., a condition presenting an enzyme capable of catalyzing the release).
  • the linkers described herein can be used, for example, to attach a cytotoxic drug payload (e.g., exatecan) to a carrier and/or a targeting moiety (e.g., nanoparticle) that binds to a cancer cell (e.g., binds to a receptor on the surface of a cancer cell) and gets internalized into the cell (e.g., through the endosome and lysosomal compartment). Once internalized, the linkers can be cleaved or degraded to release active cytotoxic drug. Specifically, the protease-cleavable linkers can release their payload under the action of proteases such as cathepsin, trypsin or other proteases in the lysosomal compartment of the cell.
  • proteases such as cathepsin, trypsin or other proteases in the lysosomal compartment of the cell.
  • the cleavable linkers described herein may comprise a structure of Formula (F): wherein each instance of [AA] is a natural or non-natural amino acid residue; z is an integer of 1 to 5; w is an integer of 1 to 4 (e.g., 2 or 3); and each denotes a point of attachment, e.g., to a spacer group (e.g., PEG) or another portion of the linker, or to an exatecan molecule.
  • F Formula
  • -[AA] w - may comprise Val-Lys, Val-Cit, Phe-Lys, Trp-Lys, Asp-Lys, Val-Arg, or Val-Ala, and z may be 2, wherein one denotes an attachment to the oxygen atom of a PEG group, and the other denotes an attachment to the nitrogen atom of exatecan.
  • -[AA] w - may comprise Val-Lys.
  • the cleavable linkers described herein may comprise a structure of Formula (F-1): wherein one denotes a point of attachment to the oxygen atom of a PEG group, and the other denotes a point of attachment to the nitrogen atom of exatecan.
  • the linkers of this disclosure can be prepared from linker precursors that contain reactive groups at one or both ends of the molecule.
  • the reactive groups can be selected to allow conjugation to exatecan or an analog thereof at one end, and also facilitate conjugation to the nanoparticle at the other end.
  • exatecan comprises a primary amine group that can facilitate its conjugation to the linker.
  • linker-payload conjugate precursors can be attached to the nanoparticle using any suitable techniques and methods, and many such techniques are well-known in the art. See, e.g., WO 2017/189961 , WO 2015/183882 , WO 2013/192609 , WO 2016/179260 and WO 2018/213851 , each of which are hereby incorporated by reference in their entireties, which describe silica-based core-shell or silica-based core nanoparticles that can be used to prepare targeted nanoparticle-based drug delivery systems. Additionally, linker-payload conjugate precursors, or ligand-linker precursors, can be attached to a nanoparticle using a reaction or method described in Kolb et al. Angew. Chem. Int. Ed. (2001) 40:2004-2021 , which is incorporated herein by reference in its entirety.
  • the linker-payload conjugate may be attached to the nanoparticle directly or indirectly through a spacer group, such as a spacer group described herein.
  • Suitable spacer groups include, but are not limited to, a divalent linker (e.g., a divalent linker described herein), such as PEG spacer, or an alkylene spacer (e.g., a methylene spacer), which may further comprise a heteroatom or cyclic group (e.g., heterocyclylene group).
  • the linker-payload conjugate can be absorbed into the interstices or pores of the silica shell, or coated onto the silica shell of the nanoparticle, such as a fluorescent nanoparticle (e.g., covalently attached to the surface of the nanoparticle).
  • the linker-payload conjugate can be associated with the fluorescent core, such as by physical absorption or by bonding interaction.
  • the linker-payload conjugate may also be associated with the PEG groups that are covalently bonded to the surface of the nanoparticle.
  • the linker-payload conjugate may be attached to the nanoparticle through the PEG.
  • the PEGs can have multiple functional groups for attachment to the nanoparticle and to the linker-payload conjugate.
  • the linker-payload conjugates may be functionalized with a hydrophilic PEG spacer.
  • the linker-payload conjugate precursor may be functionalized with a hydrophilic PEG spacer and/or suitable terminal group such as, but not limited to, an azide group, to facilitate covalently attaching the linker-payload conjugate (e.g., via the spacer group) to the surface of the nanoparticle, e.g., via reaction with a DBCO group on the nanoparticle surface).
  • terminal groups can include a nitrile oxide or nitrone, e.g., for conjugation via a 3+2 cycloaddition reaction, to a suitable group on the nanoparticle (e.g., a diene moiety).
  • the number of PEG groups as spacers that can be present in a functionalized linker-payload conjugate may range from 0 to 20, e.g., from 2 to 20, from 2 to 10, or from 5 to 8, e.g., 5, 6, 7, 8, 9, 10, 11, or 12. In preferred aspects, the number of PEG groups as spacers in a functionalized linker-payload conjugate is 9.
  • exatecan can be conjugated to a protease-cleavable linker to form the linker-payload conjugate.
  • This linker-conjugate can be prepared from a precursor functionalized with a PEG spacer that has a terminal reactive group, such as an azide, for further conjugation to the surface of the nanoparticle, e.g., via a DBCO group.
  • the protease-cleavable linker can be designed to be labile to cathepsin B (Cat-B), an enzyme that is over-expressed in malignant tumors, thereby effecting release of the cytotoxic agent, such as exatecan by a self-immolative process.
  • Cat-B cathepsin B
  • the linker payload conjugate precursor can comprise a structure of Formula (E-1): wherein y is an integer of 0 to 20, e.g., 5 to 15, e.g., 9.
  • linker and linker-payload conjugates described in the present disclosure have several advantages, ranging from superior serum stability to faster release kinetics mechanism, relative to conventional drug delivery platforms, linkers, or linker-payload conjugates. Also, the ability to pair these linkers with a variety of chemical groups provides the opportunity for the selective release of free payload/drugs, with minimal derivatization, that is a significant advantage.
  • the linker in the linker-payload conjugate is a protease-cleavable linker.
  • the NDCs of the present disclosure comprising a payload-linker moiety may comprise a structure of Formula (NP-3): (NP-3), wherein x is an integer of 0 to 10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., 4), and y is an integer of 0 to 20 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, e.g., 9), and the silicon atom is a part of the nanoparticle (e.g., bonded with the silica shell of a core-shell silica nanoparticle.
  • x may be 4, and y may be 9.
  • an NDC disclosed herein may comprise more than one molecule of Formula (NP-3), for example, the nanoparticle may comprise between about 1 and about 80 molecules of Formula (NP-3), e.g., between about 1 and about 60 molecules of Formula (NP-3), between about 1 and about 40 molecules of Formula (NP-3), between about 1 and about 30 molecules of Formula (NP-3), between about 10 and about 30 molecules of Formula (NP-3), between about 15 and about 25 molecules of Formula (NP-3), e.g., about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, or about 30 molecules of Formula (NP-3).
  • NP-3 e.g., between about 1 and about 60 molecules of Formula (NP-3), between about 1 and about 40 molecules of Formula (NP-3), between about 1 and about 30 molecules of Formula (NP-3), between about 10 and about 30 molecules of Formula (NP-3), between about 15 and about
  • an NDC of the present disclosure may undergo cleavage to release free exatecan.
  • a protease e.g., within a cancer cell, such as within the lysosome of a cancer cell
  • an NDC of the present disclosure may undergo cleavage to release free exatecan.
  • the cleavage of an NDC disclosed herein may concomitantly release exatecan, carbon dioxide, and 4-aminobenzyl alcohol from the NDC.
  • the cleavage of an exemplary NDC disclosed herein is provided in Scheme 1 below.
  • the NDCs disclosed herein may comprise both a molecule of Formula (NP-2), and a molecule of Formula (NP-3), e.g., each NDC may comprise about 1 and about 20 molecules of Formula (NP-2), and about 1 and about 30 molecules of Formula (NP-3).
  • each NDC may comprise about 10 and about 15 molecules of Formula (NP-2), and about 15 and about 25 molecules of Formula (NP-3).
  • An NDC disclosed herein may comprise an average of 13 molecules of Formula (NP-2), and an average of 21 molecules of Formula (NP-3); an average of 12 molecules of Formula (NP-2), and an average of 25 molecules of Formula (NP-3); an average of 12 molecules of Formula (NP-2), and an average of 20 molecules of Formula (NP-3).
  • compositions and methods directed to a nanoparticle-drug conjugate comprising: a nanoparticle; a targeting ligand that binds to folate receptor; and a linker-payload conjugate, wherein the NDC has an average diameter between about 1 nm and about 10 nm.
  • NDC nanoparticle-drug conjugate
  • a nanoparticle comprising folic acid as a targeting ligand, and a linker-payload conjugate comprising exatecan conjugated via a protease-cleavable linker, wherein the NDC has an average diameter between about 1 nm and about 10 nm.
  • FIG. 1 illustrates a representative nanoparticle-drug conjugate (NDC) that has an average diameter of about 6 nm, comprising a nanoparticle that comprises a silica-based core and a silica shell surrounding at least a portion of the core, polyethylene glycol (PEG) covalently bonded to the surface of the nanoparticle, and a fluorescent compound (Cy5) covalently encapsulated within the core of the nanoparticle, folic acid (FA) as the targeting ligand that can bind to a folate receptor, and a linker-payload conjugate that comprises a protease-cleavable linker-exatecan conjugate.
  • NDC nanoparticle-drug conjugate
  • folic acid is intended to encompass any amide or ester derivative of folic acid, e.g., as shown in FIG. 1 where folic acid is covalently attached to the spacer group (PEG) via an amide group.
  • the NDC may have an average diameter between about 5 nm to about 8 nm, or between about 6 nm to about 7 nm.
  • the average diameter of NDCs can be measured by any suitable methods, such as, but not limited to, fluorescence correlation spectroscopy (FCS) (see, e.g., FIG. 6 ) and gel permeation chromatography (GPC) ( FIG. 7 ).
  • FCS fluorescence correlation spectroscopy
  • GPS gel permeation chromatography
  • the NDCs of the present disclosure can comprise nanoparticles that can be functionalized with contrast agents for positron emission tomography (PET), single photon emission computed tomography (SPECT), computerized tomography (CT), magnetic resonance imaging (MRI), and optical imaging (such as fluorescence imaging including near-infrared fluorescence (NIRF) imaging, bio luminescence imaging, or combinations thereof).
  • PET positron emission tomography
  • SPECT single photon emission computed tomography
  • CT computerized tomography
  • MRI magnetic resonance imaging
  • optical imaging such as fluorescence imaging including near-infrared fluorescence (NIRF) imaging, bio luminescence imaging, or combinations thereof.
  • a contrast agent such as a radionuclide (radiolabel) including, but not limited to 89 Zr, 64 Cu, 68 Ga, 86 Y, 124 I and 177 Lu, may be attached to the nanoparticle.
  • the nanoparticle can be attached to a chelator moiety, for example, DFO, DOTA, TETA and DTPA, that is adapted to bind a radionuclide.
  • a chelator moiety for example, DFO, DOTA, TETA and DTPA
  • Such nanoparticle may be detected by PET, SPECT, CT, MRI, or optical imaging (such as fluorescence imaging including near-infrared fluorescence (NIRF) imaging, bio luminescence imaging, or combinations thereof).
  • NIRF near-infrared fluorescence
  • the radionuclide can additionally serve as a therapeutic agent for creating a multitherapeutic platform. This coupling allows the therapeutic agent to be delivered to the specific cell type through the specific binding between the targeting ligand and the cellular component.
  • a linker-payload conjugate may comprise a compound of Formula (I) or a salt thereof, wherein, line represents an attachment to the nanoparticle through a spacer group;
  • A is a dipeptide selected from the group consisting of Val-Cit, Phe-Lys, Trp-Lys, Asp-Lys, Val-Lys, Val-Arg, and Val-Ala, or
  • A is a tetrapeptide selected from the group consisting of Val-Phe-Gly-Sar, Val-Cit-Gly-Sar, Val-Lys-Gly-Sar, Val-Ala-Gly-Sar, Val-Phe-Gly-Pro, Val-Cit-Gly-Pro, Val-Lys-Gly-Pro, Val-Ala-Gly-Pro, Val-Cit-Gly-any natural or unnatural N-alkyl substituted alpha amino acid, Val-Lys-Gly-any natural or unnatural N-alkyl substituted al
  • A is Val-Lys; R 1 -R 5 are each independently hydrogen; X is absent; Y is wherein the carbonyl in is bonded to Z; n is 1; X 1 , X 2 , X 3 , and X 4 are each independently -CH-; Z is-NR c - wherein R c is hydrogen, and wherein the N is the nitrogen atom present in the exatecan payload.
  • the payload may be exatecan, which has a functional group that is bonded to the linker, wherein the functional group is an amine (when exatecan is bonded to the linker, it is a secondary amine, and once released (or prior to conjugation), i.e., as a separate molecular entity, the amine of exatecan is a primary amine).
  • Linker-payload conjugates of the present disclosure include, but are not limited to the following substructures, wherein line represents a direct bond to the nanoparticle or an indirect bond to the nanoparticle through a spacer group.
  • Suitable spacer groups include, but are not limited to a PEG spacer, or an alkylene spacer (e.g., methylene spacer), which may further comprise heteroatoms, or cyclic groups (e.g., heterocyclylene groups).
  • the spacer group is a PEG spacer.
  • An exemplary linker-payload conjugate of Formula (I) of the present disclosure includes the following sub-structure:
  • the linkers of this disclosure, and/or their precursors can contain reactive groups at both ends of the molecule.
  • the reactive groups can be selected to allow conjugation to exatecan or a salt or analog thereof at one end, and also facilitate conjugation to a nanoparticle (e.g., via a spacer group) at the other end.
  • the linker can connect to exatecan via a chemically reactive functional group that is a part of the exatecan, such as the primary amine of exatecan (that becomes a secondary amine upon conjugation to the linker).
  • the linker can be conjugated to a functionalized polyethylene glycol or a C 5 -C 6 alkyl chain via a chemically reactive functional group that is a part of the linker such as a primary or secondary amine or carboxyl group.
  • Protease-cleavable Linkers Proteases are involved in all stages of cancer disease from tumor cells growth and survival, to angiogenesis and invasions. Therefore, they can be utilized to treat cancer as selective triggers towards activation of linker/payload system.
  • This disclosure relates to linkers that are cleavable by the action of proteases thereby releasing the free payload (e.g., exatecan). Lysosomal proteases such as cathepsin B and serine proteases such as cathepsin A or tripeptidyl-peptidase I have been extensively studied in the context of prodrug development.
  • Proteolytic enzymes such as caspases are also well-known to be utilized as biological triggers for the selective activation of payload or for specific cargo delivery to a target cell such as a cancer cell.
  • a linker (or precursor thereof) can comprise a compound of Formula (I-A) wherein: A is a dipeptide selected from the group consisting of Val-Cit, Phe-Lys, Trp-Lys, Asp-Lys, Val-Lys, Val-Arg, and Val-Ala, or A is a tetrapeptide selected from the group consisting of Val-Phe-Gly-Sar, Val-Cit-Gly-Sar, Val-Lys-Gly-Sar, Val-Ala-Gly-Sar, Val-Phe-Gly-Pro, Val-Cit-Gly-Pro, Val-Lys-Gly-Pro, Val-Ala-Gly-Pro, Val-Cit-Gly-any natural or unnatural N-alkyl substituted alpha amino acid, Val-Lys-Gly-any natural or unnatural N-alkyl substituted alpha amino acid, Val-Phe-Gly-any natural or unnatural N-alkyl substituted
  • A is Val-Lys; R 1 -R 5 are each independently hydrogen; X is absent; Y is wherein the carbonyl in is bonded to Z; n is 1; X 1 , X 2 , X 3 and X 4 are each independently -CH-; Z 1 is a functional group selected from the group consisting of halo, hydroxy, -OSO 2 -CH 3 , -OSO 2 CF 3 , 4-nitrophenoxy, -COCl, and -COOH; Z 2 is a functional group selected from the group consisting of -NH 2 , -NHR c , and -COOH or Z 2 is -C(O)-T 1 , wherein T 1 is as defined in Formula (I-A).
  • the present disclosure further provides a pharmaceutical composition for treating a disease (e.g., cancer, such as a cancer associated with folate receptor expressing tumor), wherein the composition comprises an effective amount of an NDC described herein.
  • a disease e.g., cancer, such as a cancer associated with folate receptor expressing tumor
  • the composition comprises an effective amount of an NDC described herein.
  • the pharmaceutical composition comprising the NDCs can be used to treat cancer selected from the group consisting of ovarian cancer, endometrial cancer, fallopian tube cancer, cervical cancer, breast cancer, lung cancer, mesothelioma, uterine cancer, gastrointestinal cancer (e.g., esophageal cancer, colon cancer, rectal cancer, and stomach cancer), pancreatic cancer, bladder cancer, kidney cancer, liver cancer, head and neck cancer, brain cancer, thyroid cancer, skin cancer, prostate cancer, testicular cancer, acute myeloid leukemia (AML, e.g., pediatric AML), and chronic myelogenous leukemia (CML).
  • the pharmaceutical composition comprising the NDCs may also be used for targeting tumor associated macrophages, e.g., to modify the immune status of a tumor in a subject.
  • compositions of the present disclosure may comprise a pharmaceutically acceptable excipient, such as a non-toxic carrier, adjuvant, diluent, or vehicle that does not negatively impact the pharmacological activity of the NDCs with which it is formulated.
  • a pharmaceutically acceptable excipient such as a non-toxic carrier, adjuvant, diluent, or vehicle that does not negatively impact the pharmacological activity of the NDCs with which it is formulated.
  • Pharmaceutically acceptable excipients useful in the manufacture of the pharmaceutical compositions of the present disclosure are any of those that are well known in the art of pharmaceutical formulation, and can include inert diluents, dispersing and/or granulating agents, surface active agents and/or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and/or oils.
  • compositions of the present disclosure include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (e.g., human serum albumin), buffer substances (e.g., phosphates), glycine, sorbic acid, potassium sorbate, glyceride mixtures (e.g., mixtures of saturated vegetable fatty acids), water, salts or electrolytes (e.g., protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts), colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and wool fat.
  • compositions of the present disclosure may be administered orally in the form of a suitable pharmaceutical unit dosage form.
  • the pharmaceutical compositions of the present disclosure may be prepared in many forms that include tablets, hard or soft gelatin capsules, aqueous solutions, suspensions, liposomes, and other slow-release formulations, such as shaped polymeric gels.
  • Suitable modes of administration for the NDCs or composition include, but are not limited to, oral, intravenous, rectal, sublingual, mucosal, nasal, ophthalmic, subcutaneous, intramuscular, transdermal, spinal, intrathecal, intra-articular, intra-arterial, sub-arachnoid, bronchial, lymphatic administration, intra-tumoral, and other routes suitable for systemic delivery of active ingredients.
  • the present pharmaceutical composition may be administered by any method known in the art, including, without limitation, transdermal (passive via patch, gel, cream, ointment or iontophoretic); intravenous (bolus, infusion); subcutaneous (infusion, depot); transmucosal (buccal and sublingual, e.g., orodispersible tablets, wafers, film, and effervescent formulations); conjunctival (eyedrops); rectal (suppository, enema)); or intradermal (bolus, infusion, depot).
  • transdermal passive via patch, gel, cream, ointment or iontophoretic
  • intravenous bolus, infusion
  • subcutaneous infusion, depot
  • transmucosal bilingual and sublingual, e.g., orodispersible tablets, wafers, film, and effervescent formulations
  • conjunctival eyedrops
  • rectal rectal
  • intradermal bolus, infusion, depot
  • Oral liquid pharmaceutical compositions may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for constitution with water or other suitable vehicle before use.
  • Such liquid pharmaceutical compositions may contain conventional additives such as suspending agents, emulsifying agents, non-aqueous vehicles (which may include edible oils), or preservatives.
  • compositions of the present disclosure may also be formulated for parenteral administration (e.g., by injection, for example, bolus injection or continuous infusion) and may be presented in unit dosage form in ampules, pre-filled syringes, infusion containers (e.g., small volume infusion containers), or multi-dose containers, that may contain an added preservative.
  • parenteral administration e.g., by injection, for example, bolus injection or continuous infusion
  • infusion containers e.g., small volume infusion containers
  • multi-dose containers that may contain an added preservative.
  • compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulating agents such as suspending, stabilizing and/or dispersing agents.
  • the pharmaceutical compositions of the present disclosure may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilization from solution, for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use.
  • the pharmaceutical compositions may be formulated as an ointment, cream, or lotion, or as the active ingredient of a transdermal patch.
  • Suitable transdermal delivery systems are disclosed, for example, in A. Fisher et al. (U.S. Pat. No. 4,788,603 ), and R. Bawa et al. (U.S. Pat. Nos. 4,931,279 ; 4,668,506 ; and 4,713,224 ), which are incorporated herein by reference in their entireties.
  • Ointments and creams may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents.
  • Lotions may be formulated with an aqueous or oily base and will in general also contain one or more emulsifying agents, stabilizing agents, dispersing agents, suspending agents, thickening agents, or coloring agents.
  • the pharmaceutical compositions can also be delivered via ionophoresis, e.g., as disclosed in U.S. Pat. Nos. 4,140,122 ; 4,383,529 ; or 4,051,842 , each of which are incorporated herein by reference in their entireties.
  • compositions suitable for topical administration in the mouth include unit dosage forms such as lozenges comprising a pharmaceutical composition of the present disclosure in a flavored base, such as sucrose and acacia or tragacanth; pastilles comprising the pharmaceutical composition in an inert base such as gelatin and glycerin or sucrose and acacia; mucoadherent gels, and mouthwashes comprising the pharmaceutical composition in a suitable liquid carrier.
  • unit dosage forms such as lozenges comprising a pharmaceutical composition of the present disclosure in a flavored base, such as sucrose and acacia or tragacanth
  • pastilles comprising the pharmaceutical composition in an inert base such as gelatin and glycerin or sucrose and acacia
  • mucoadherent gels such as mucoadherent gels
  • mouthwashes comprising the pharmaceutical composition in a suitable liquid carrier.
  • the pharmaceutical compositions can be administered as drops, gels ( S. Chrai et al, U.S. Pat. No. 4,255,415 ), gums ( S. L. Lin et al, U.S. Pat. No. 4,136,177 ) or via a prolonged-release ocular insert (A. S. Michaels, U.S. Pat. No. 3,867,519 and H. M. Haddad et al., U.S. Pat. No. 3,870,791 ), each of which are incorporated herein by reference in their entireties.
  • compositions can be adapted to give sustained release of a therapeutic compound employed, e.g., by combination with certain hydrophilic polymer matrices, e.g., comprising natural gels, synthetic polymer gels or mixtures thereof.
  • compositions suitable for rectal administration wherein the carrier is a solid are most preferably presented as unit dose suppositories.
  • Suitable carriers include cocoa butter and other materials commonly used in the art, and the suppositories may be conveniently formed by admixture of the pharmaceutical composition with the softened or melted carrier(s) followed by chilling and shaping in molds.
  • compositions suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams, or sprays containing, in addition to the nanoparticles and the therapeutic agent, a carrier.
  • a carrier such carriers are well known in the art.
  • the pharmaceutical compositions according to the present disclosure are conveniently delivered from an insufflator, nebulizer or a pressurized pack or other convenient means of delivering an aerosol spray.
  • Pressurized packs may comprise a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • the pharmaceutical compositions of the present disclosure may take the form of a dry powder composition, for example, a powder mix of the pharmaceutical composition and a suitable powder base such as lactose or starch.
  • the powder composition may be presented in unit dosage form in, for example, capsules or cartridges or, e.g., gelatin or blister packs from which the powder may be administered with the aid of an inhalator or insufflator.
  • compositions of the present disclosure may be administered via a liquid spray, such as via a plastic bottle atomizer.
  • a liquid spray such as via a plastic bottle atomizer.
  • MISTOMETER ® isoproterenol inhaler- Wintrop
  • MEDIHALER ® isoproterenol inhaler-Riker
  • compositions of the present disclosure may also contain other adjuvants such as flavorings, colorings, anti-microbial agents, or preservatives.
  • the amount of the pharmaceutical compositions suitable for use in treatment will vary not only with the therapeutic agent selected but also with the route of administration, the nature of the condition being treated and the age and condition of the patient and will be ultimately at the discretion of the attendant physician or clinician.
  • physicians' Desk Reference Charles E. Baker, Jr., Pub., Medical Economics Co., Oradell, N.J. (41st ed., 1987 ), each of which are incorporated herein by reference in their entireties.
  • NDCs of the present disclosure can be administered to a subject.
  • the subject can be a mammal, preferably a human.
  • Mammals include, but are not limited to, murines, rats, rabbits, simians, bovines, ovine, swine, canines, feline, farm animals, sport animals, pets, equine, and primates.
  • NDCs may be administered to a subject by, but not restricted to, the following routes: oral, intravenous, nasal, subcutaneous, local, intramuscular or transdermal.
  • the NDCs of the present disclosure may be administered to a subject intravenously.
  • the methods and compositions of the present disclosure can be used to help a physician or surgeon to identify and characterize areas of disease, such as cancers, including, but not restricted to, cancers that overexpress FR, to distinguish diseased and normal tissue, such as detecting tumor margins that are difficult to detect using an ordinary operating microscope, e.g., in brain surgery, to help dictate a therapeutic or surgical intervention, e.g., by determining whether a lesion is cancerous and should be removed or non-cancerous and left alone, or in surgically staging a disease.
  • areas of disease such as cancers, including, but not restricted to, cancers that overexpress FR
  • detecting tumor margins that are difficult to detect using an ordinary operating microscope, e.g., in brain surgery
  • a therapeutic or surgical intervention e.g., by determining whether a lesion is cancerous and should be removed or non-cancerous and left alone, or in surgically staging a disease.
  • compositions of the present disclosure may be used, but are not limited to, metastatic disease detection, treatment response monitoring, and targeted delivery of payload, including by passing the blood-brain barrier.
  • the methods and compositions of the present disclosure can also be used in the detection, characterization and/or determination of the localization of a disease, including early disease, the severity of a disease or a disease-associated condition, the staging of a disease, and/or monitoring a disease.
  • the presence, absence, or level of an emitted signal can be indicative of a disease state.
  • the methods and compositions of the present disclosure can also be used to monitor and/or guide various therapeutic interventions, such as surgical and catheter-based procedures, and monitoring drug therapy, including cell based therapies.
  • the methods of the present disclosure can also be used in prognosis of a disease or disease condition.
  • Cellular subpopulations residing within or marginating the disease site such as stem-like cells (“cancer stem cells”) and/or inflammatory/phagocytic cells may be identified and characterized using the methods and compositions of the present disclosure.
  • examples of such disease or disease conditions that can be detected or monitored include cancer (for example, melanoma, thyroid, colorectal, ovarian, lung, breast, prostate, cervical, skin, brain, gastrointestinal, mouth, kidney, esophageal, bone cancer), that can be used to identify subjects that have an increased susceptibility for developing cancer and/or malignancies, i.e., they are predisposed to develop cancer and/or malignancies, inflammation (for example, inflammatory conditions induced by the presence of cancerous lesions), cardiovascular disease (for example, atherosclerosis and inflammatory conditions of blood vessels, ischemia, stroke, thrombosis), dermatologic disease (for example, Kaposi's Sarcoma, psoriasis), ophthalmic disease (for example, macular degeneration, diabetic retinopathy), infectious disease (for example, bacterial, viral, fungal and parasitic infections, including Acquired Immunodeficiency Syndrome (AIDS)), immunologic
  • cancer for example, melanoma, thyroid, colorec
  • the methods and compositions of the present disclosure can be used, for example, to determine the presence and/or localization of tumor and/or co-resident stem-like cells ("cancer stem cells"), the presence and/or localization of inflammatory cells, including the presence of activated macrophages, for instance in peritumoral regions, the presence and in localization of vascular disease including areas at risk for acute occlusion (i.e., vulnerable plaques) in coronary and peripheral arteries, regions of expanding aneurysms, unstable plaque in carotid arteries, and ischemic areas.
  • the methods and compositions of the present disclosure can also be used in identification and evaluation of cell death, injury, apoptosis, necrosis, hypoxia and angiogenesis ( PCT/US2006/049222 ).
  • the methods of the present disclosure comprise administering to a subject in need thereof an effective amount of an NDC described herein.
  • the NDC can be administered to the subject in need thereof intravenously.
  • An "effective amount" is an amount of the NDC that elicits a desired biological or medicinal response under the conditions of administration, such as an amount that reduces the signs and/or symptoms of a disease or disorder being treated, e.g., reduces tumor size or tumor burden.
  • the actual amount administered can be determined by an ordinarily skilled clinician based upon, for example, the subject's age, weight, sex, general heath and tolerance to drugs, severity of disease, dosage form selected, route of administration, and other factors.
  • the subject has a cancer selected from the group consisting of ovarian cancer, endometrial cancer, fallopian tube cancer, cervical cancer, breast cancer, lung cancer, mesothelioma, uterine cancer, gastrointestinal cancer (e.g., esophageal cancer, colon cancer, rectal cancer, and stomach cancer), pancreatic cancer, bladder cancer, kidney cancer, liver cancer, head and neck cancer, brain cancer, thyroid cancer, skin cancer, prostate cancer, testicular cancer, acute myeloid leukemia (AML, e.g., pediatric AML), and chronic myelogenous leukemia (CML).
  • AML acute myeloid leukemia
  • CML chronic myelogenous leukemia
  • the present disclosure also includes use of NDCs for treating a folate receptor expressing tumor.
  • the use of NDC may comprise administration to the subject in need thereof intravenously.
  • the present disclosure also relates to the use of NDCs in a subject with cancer selected from the group consisting of ovarian cancer, endometrial cancer, fallopian tube cancer, cervical cancer, breast cancer, lung cancer, mesothelioma, uterine cancer, gastrointestinal cancer (e.g., esophageal cancer, colon cancer, rectal cancer, and stomach cancer), pancreatic cancer, bladder cancer, kidney cancer, liver cancer, head and neck cancer, brain cancer, thyroid cancer, skin cancer, prostate cancer, testicular cancer, acute myeloid leukemia (AML, e.g., pediatric AML), and chronic myelogenous leukemia (CML).
  • cancer selected from the group consisting of ovarian cancer, endometrial cancer, fallopian tube cancer, cervical cancer, breast cancer, lung cancer, mesothelioma, uterine cancer, gastrointestinal cancer (e.g., esophageal cancer, colon cancer, rectal cancer, and stomach cancer), pancreatic cancer, bladder cancer, kidney cancer,
  • the NDCs of the present disclosure may also be used in the manufacture of a medicament for treating a folate receptor expressing tumor, wherein the NDC is administered to the subject in need thereof intravenously and wherein the subject has a cancer selected from the group consisting of ovarian cancer, endometrial cancer, fallopian tube cancer, cervical cancer, breast cancer, lung cancer, mesothelioma, uterine cancer, gastrointestinal cancer (e.g., esophageal cancer, colon cancer, rectal cancer, and stomach cancer), pancreatic cancer, bladder cancer, kidney cancer, liver cancer, head and neck cancer, brain cancer, thyroid cancer, skin cancer, prostate cancer, testicular cancer, acute myeloid leukemia (AML, e.g., pediatric AML), and chronic myelogenous leukemia (CML).
  • AML acute myeloid leukemia
  • CML chronic myelogenous leukemia
  • compositions and methods disclosed herein can include compositions and methods that include administering a NDC as disclosed herein in combination with one or more additional anti-cancer agents.
  • the NDC can be administered before, substantially concurrently with, or after the additional agent or agents.
  • additional agents include, for example chemotherapeutic agents such as mechlorethamine, cyclophosphamide, melphalan, chlorambucil, ifosfamide, busulfan, N-nitroso-N-methylurea, carmustine, lomustine, semustine, fotemustine, streptozotocin, dacarbazine, mitozolomide, temozolomide, thiotepa, mitomycin, diaziquone, cisplatin, carboplatin, oxaliplatin, procarbazine, hexamethylmelamine, methotrexate, pemetrexed, fluorouracil (e.g.
  • 5-fluorouracil capecitabine, cytarabine, gemcitabine, decitabine, azacitidine, fludarabine, nelarabine, cladribine, clofarabine, pentostatin, thioguanine, mercaptopurine, vincristine, vinblastine, vinorelbine, vindesine, vinflunine, paclitaxel, docetaxel, irinotecan, topotecan, camptothecin, etoposide, mitoxantrone,teniposide, novobiocin, merbarone, doxorubicin, daunorubicin, epirubicin, idarubicin, pirarubicin, aclarubicin, mitomycin C, actinomycin, bleomycin, bisantrene, gemcitabine, cytarabine, and the like.
  • anti-cancer agents that can be used with a NDC in the compositions and methods disclosed herein include, immune check point inhibitors (e.g., anti-PD1, anti-PDL1, anti-CTLA4 antibodies), hormone receptor antagonists, other chemotherapeutic conjugates (e.g., in the form of antibody-drug conjugates, nanoparticle drug conjugates, and the like), and the like.
  • immune check point inhibitors e.g., anti-PD1, anti-PDL1, anti-CTLA4 antibodies
  • hormone receptor antagonists e.g., other chemotherapeutic conjugates (e.g., in the form of antibody-drug conjugates, nanoparticle drug conjugates, and the like), and the like.
  • alkyl refers to monovalent aliphatic hydrocarbon group that may comprise 1 to 18 carbon atoms, such as 1 to about 12 carbon atoms, or 1 to about 6 carbon atoms (“C 1-18 alkyl”).
  • An alkyl group can be straight chain, branched chain, monocyclic moiety or polycyclic moiety or combinations thereof.
  • alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, iso -butyl, sec -butyl, tert -butyl, pentyl, hexyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornyl, and the like.
  • Each instance of an alkyl group may be independently optionally substituted, i.e., unsubstituted (an "unsubstituted alkyl") or substituted (a "substituted alkyl") with one or more substituents e.g., for instance from 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.
  • alkenyl refers to a monovalent straight-chain or branched hydrocarbon group having from 2 to 18 carbon atoms, one or more carbon-carbon double bonds, and no triple bonds ("C 2-18 alkenyl").
  • An alkenyl group may have 2 to 8 carbon atoms, 2 to 6 carbon atoms, 2 to 5 carbon atoms, 2 to 4 carbon atoms, or 2 to 3 carbon atoms.
  • the one or more carbon-carbon double bonds can be internal (such as in 2-butenyl) or terminal (such as in 1-butenyl).
  • alkenyl groups include ethenyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, butadienyl, pentenyl, pentadienyl, hexenyl, heptenyl, octenyl, octatrienyl, and the like.
  • Each instance of an alkenyl group may be independently optionally substituted, i.e., unsubstituted (an "unsubstituted alkenyl") or substituted (a "substituted alkenyl") with one or more substituents e.g., for instance from 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.
  • alkynyl refers to a monovalent straight-chain or branched hydrocarbon group having from 2 to 18 carbon atoms, one or more carbon-carbon triple bonds ("C 2-18 alkynyl").
  • the alkynyl group may have 2 to 8 carbon atoms, 2 to 6 carbon atoms, 2 to 5 carbon atoms, 2 to 4 carbon atoms, or 2 to 3 carbon atoms.
  • the one or more carbon-carbon triple bonds can be internal (such as in 2-butynyl) or terminal (such as in 1-butynyl).
  • alkynyl groups include ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, and the like.
  • Each instance of an alkynyl group may be independently optionally substituted, i.e., unsubstituted (an "unsubstituted alkynyl") or substituted (a "substituted alkynyl") with one or more substituents, e.g., for instance from 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.
  • heteroalkyl refers to a non-cyclic stable straight or branched chain, or combinations thereof, including at least one carbon atom and at least one heteroatom selected from the group consisting of O, N, P, Si, and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized.
  • the heteroatom(s) O, N, P, S, and Si may be placed at any position of the heteroalkyl group.
  • alkylene alkenylene, alkynylene, or “heteroalkylene,” alone or as part of another substituent, mean, unless otherwise stated, a divalent radical derived from an alkyl, alkenyl, alkynyl, or heteroalkyl, respectively.
  • alkenylene by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from an alkene.
  • alkylene, alkenylene, alkynylene, or heteroalkylene group may be described as, e.g., a C 1-6 -membered alkylene, C 1-6 -membered alkenylene, C 1-6 -membered alkynylene, or C 1-6 -membered heteroalkylene, wherein the term “membered” refers to the non-hydrogen atoms within the moiety.
  • heteroatoms can also occupy either or both of the chain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, and the like).
  • alkylene and heteroalkylene linking groups no orientation of the linking group is implied by the direction in which the formula of the linking group is written.
  • the formula -C(O) 2 R'- may represent both -C(O) 2 R'- and -R'C(O) 2 -.
  • Each instance of an alkylene, alkenylene, alkynylene, or heteroalkylene group may be independently optionally substituted, i.e., unsubstituted (an "unsubstituted alkylene") or substituted (a "substituted heteroalkylene") with one or more substituents.
  • substituted alkyl refers to alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl moieties, respectively, having substituents replacing one or more hydrogen atoms on one or more carbons or heteroatoms of the moiety.
  • substituents can include, for example, alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, amino (including alkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sul
  • alkoxy refers to a group of formula -O-alkyl.
  • alkoxy or “alkoxyl” includes substituted and unsubstituted alkyl, alkenyl and alkynyl groups covalently linked to an oxygen atom.
  • alkoxy groups or alkoxyl radicals include, but are not limited to, methoxy, ethoxy, isopropyloxy, propoxy, butoxy and pentoxy groups.
  • substituted alkoxy groups include halogenated alkoxy groups.
  • the alkoxy groups can be substituted with groups such as alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, s
  • aryl refers to stable aromatic ring system, that may be monocyclic or polycyclic, of which all the ring atoms are carbon, and which may be substituted or unsubstituted.
  • the aromatic ring system may have, for example, 3-7 ring atoms. Examples include phenyl, benzyl, naphthyl, anthracyl, and the like.
  • Each instance of an aryl group may be independently optionally substituted, i.e., unsubstituted (an "unsubstituted aryl") or substituted (a "substituted aryl") with one or more substituents.
  • heteroaryl refers to an aryl group that includes one or more ring heteroatoms.
  • a heteroaryl can include a stable 5-, 6-, or 7-membered monocyclic or 7-, 8-, or 9-membered bicyclic aromatic heterocyclic ring which consists of carbon atoms and one or more heteroatoms, independently selected from the group consisting of nitrogen, oxygen and sulfur.
  • the nitrogen atom may be substituted or unsubstituted (e.g., N or NR 4 wherein R 4 is H or other substituents, as defined).
  • heteroaryl groups include pyrrole, furan, indole, thiophene, thiazole, isothiazole, imidazole, triazole, tetrazole, pyrazole, oxazole, isoxazole, pyridine, pyrazine, pyridazine, pyrimidine, and the like.
  • cycloalkylene As used herein, the terms “cycloalkylene,” “heterocyclylene,” “arylene,” and “heteroarylene,” alone or as part of another substituent, mean a divalent radical derived from a cycloalkyl, heterocyclyl, aryl, and heteroaryl, respectively.
  • Each instance of a cycloalkylene, heterocyclylene, arylene, or heteroarylene may be independently optionally substituted, i.e., unsubstituted (an “unsubstituted arylene”) or substituted (a "substituted heteroarylene”) with one or more substituents.
  • cycloalkyl is intended to include non-aromatic cyclic hydrocarbon rings, such as hydrocarbon rings having from three to eight carbon atoms in their ring structure.
  • Cycloalkyl can include cyclobutyl, cyclopropyl, cyclopentyl, cyclohexyl and the like.
  • the cycloalkyl group can be either monocyclic (“monocyclic cycloalkyl”) or contain a fused, bridged or spiro ring system such as a bicyclic system (“bicyclic cycloalkyl”) and can be saturated or can be partially unsaturated.
  • Cycloalkyl also includes ring systems wherein the cycloalkyl ring, as defined above, is fused with one or more aryl groups wherein the point of attachment is on the cycloalkyl ring, and in such instances, the number of carbons continue to designate the number of carbons in the cycloalkyl ring system.
  • Each instance of a cycloalkyl group may be independently optionally substituted, i.e., unsubstituted (an "unsubstituted cycloalkyl") or substituted (a "substituted cycloalkyl") with one or more substituents.
  • heterocyclyl refers to a monovalent cyclic molecular structure comprising atoms of at least two different elements in the ring or rings (i.e., a radical of a heterocyclic ring). Additional reference is made to: Oxford Dictionary of Biochemistry and Molecular Biology, Oxford University Press, Oxford, 1997 as evidence that heterocyclic ring is a term well-established in field of organic chemistry.
  • dipeptide refers to a peptide that is composed of two amino-acid residues, that may be denoted herein as -A 1 -A 2 -.
  • dipeptides employed in the synthesis of protease-cleavable linker-payload conjugates of the present disclosure may be selected from the group consisting of Val-Cit, Phe-Lys, Trp-Lys, Asp-Lys, Val-Lys, and Val-Ala.
  • a functionalized polyethylene glycol refers to the polyethylene glycol comprising a functional group.
  • a functionalized polyethylene glycol may be polyethylene glycol functionalized with a terminal group selected from the group consisting of azide, and wherein R 1' , R 2' , R 3' , R 4' and R 5' in each occurrence is independently hydrogen, substituted or unsubstituted C 1-6 alkyl or substituted or unsubstituted C 1-6 cycloalkyl.
  • R 1' , R 2' , R 3' , R 4' and R 5' in each occurrence is hydrogen.
  • R 1' , R 2' , R 3' , R 4' and R 5' in each occurrence is methyl.
  • the term “functionalized polyethylene glycol” refers to, but is not limited to the following structures.
  • Ti may refer to a functionalized polyethylene glycol or a C 5 -C 6 alkyl chain that has a terminal group selected from the group consisting of azide, wherein R 1' , R 2' , R 3' , R 4' and R 5' in each occurrence is independently hydrogen, substituted or unsubstituted C 1-6 alkyl or substituted or unsubstituted C 1-6 cycloalkyl.
  • R 1' , R 2' , R 3' , R 4' and R 5' in each occurrence is hydrogen.
  • T 1 , R 1' , R 2' , R 3' , R 4' and R 5' in each occurrence is methyl.
  • Ti is a functionalized polyethylene glycol that has an azide terminal group.
  • Ti is a C 5 -C 6 alkyl chain that has an azide terminal group.
  • the repeat unit (-O-CH 2 -CH 2 -) of polyethylene glycol (PEG) can range from 5-20 units, preferably 5-15 units and more preferably 6-12.
  • Ti may refer to a C 5 -C 6 alkyl chain that has a terminal group selected from the group consisting of azide, and wherein R 1' , R 2' , R 3' , R 4' and R 5' in each occurrence is independently hydrogen, substituted or unsubstituted C 1-6 alkyl or substituted or unsubstituted C 1-6 cycloalkyl.
  • R 1' , R 2' , R 3' , R 4' and R 5' in each occurrence is hydrogen.
  • R 1' , R 2' , R 3' , R 4' and R 5' in each occurrence is methyl.
  • Monofunctionalized azide-terminated PEG and monofunctionalized azide-terminated C 5 -C 6 alkyl chain can be made from PEG using known procedures and suitable reagents, such as those disclosed in the Schemes provided herein.
  • halo or halogen refers to F, Cl, Br, or I.
  • An aryl or heteroaryl group described herein can be substituted at one or more ring positions with such substituents as described above, for example, alkyl, alkenyl, akynyl, halogen, hydroxyl, alkoxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkylaminocarbonyl, aralkylaminocarbonyl, alkenylaminocarbonyl, alkylcarbonyl, arylcarbonyl, aralkylcarbonyl, alkenylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, phosphate, phosphonato, phosphinato, amino (including alkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino), acylamino (including alkylcarbonylamin
  • hydroxy refers to a group of formula -OH.
  • hydroxyl refers to a hydroxyl radical (.OH).
  • the phrase "optionally substituted” means unsubstituted or substituted.
  • substituted means that at least one hydrogen present on a group (e.g., a carbon or nitrogen atom) is replaced with a permissible substituent, e.g., a substituent which upon substitution results in a stable compound.
  • a permissible substituent e.g., a substituent which upon substitution results in a stable compound.
  • substituted can include substitution with all permissible substituents of organic compounds, such as any of the substituents described herein that result in the formation of a stable compound.
  • heteroatoms such as nitrogen may have hydrogen substituents and/or any suitable substituent as described herein which satisfy the valencies of the heteroatoms and results in the formation of a stable moiety.
  • Tetrapeptide refers to a peptide that is composed of four amino-acid residues, that may be denoted herein as -A 1 -A 2 -A 3 -A 4 -.
  • Tetrapeptides employed in the synthesis of protease-cleavable linker-payload conjugates of the present disclosure is selected from the group consisting of Val-Phe-Gly-Sar, Val-Cit-Gly-Sar, Val-Lys-Gly-Sar, Val-Ala-Gly-Sar, Val-Phe-Gly-Pro, Val-Cit-Gly-Pro, Val-Lys-Gly-Pro, Val-Ala-Gly-Pro, Val-Cit-Gly-any natural or unnatural N-alkyl substituted alpha amino acid, Val-Lys-Gly-any natural or unnatural N-alkyl substituted alpha amino acid, Val-Phe-Gly-any natural or unnatural N-alkyl substituted alpha amino acid
  • protecting group refers to a particular functional moiety, e.g., O, S, or N, that is temporarily blocked so that a reaction can be carried out selectively at another reactive site in a multifunctional compound.
  • Protecting groups may be introduced and removed at appropriate stages during the synthesis of a compound using methods that are known to one of ordinary skill in the art. The protecting groups are applied according to standard methods of organic synthesis as described in the literature ( Theodora W. Greene and Peter G. M. Wuts (2007) Protecting Groups in Organic Synthesis, 4th edition, John Wiley and Sons , incorporated by reference with respect to protecting groups).
  • oxygen protecting groups include, but are not limited to, oxygen, sulfur, nitrogen and carbon protecting groups.
  • oxygen protecting groups include, but are not limited to, methyl ethers, substituted methyl ethers (e.g., MOM (methoxymethyl ether), MTM (methylthiomethyl ether), BOM (benzyloxymethyl ether), PMBM (pimethoxybenzyloxymethyl ether), optionally substituted ethyl ethers, optionally substituted benzyl ethers, silyl ethers (e.g., TMS (trimethylsilyl ether), TES (triethylsilylether), TIPS (triisopropylsilyl ether), TBDMS (t-butyldimethylsilyl ether), tribenzyl silyl ether, TBDPS (t-butyldiphenyl silyl ether), esters (e.g., formate, acetate, benzoate (Bz), trifluoroacetate, dichlor
  • nitrogen protecting groups include, but are not limited to, carbamates (including methyl, ethyl and substituted ethyl carbamates (e.g., Troc), amides, cyclic imide derivatives, N-Alkyl and N-Aryl amines, imine derivatives, and enamine derivatives, etc.
  • Amino protecting groups include, but are not limited to fluorenylmethyloxycarbonyl (Fmoc), tert-butyloxycarbonyl (Boc), carboxybenzyl (Cbz), acetamide, trifluoroacetamide, etc. Certain other exemplary protecting groups are detailed herein, however, it will be appreciated that the present disclosure is not intended to be limited to these protecting groups; rather, a variety of additional equivalent protecting groups may be utilized according to methods known to one skilled in the art.
  • a nanoparticle-drug-conjugate may sometimes be referred to as a CDC (C'Dot-drug-conjugate), e.g., a FA-CDC.
  • protecting groups may be necessary to prevent certain functional groups from undergoing undesired reactions.
  • suitable protecting group for a particular functional group as well as suitable conditions for protection and deprotection are well known in the art. For example, numerous protecting groups, and their introduction and removal, are described in Greene et al. Protecting Groups in Organic Synthesis, Second Edition, Wiley, New York, 1991 , and references cited therein.
  • Reagents were purchased from commercial suppliers (Combi-Blocks/SIGMA-ALDRICH) and used without further purification. All non-aqueous reactions were run in flame-dried glassware under a positive pressure of argon. Anhydrous solvents were purchased from commercial suppliers (RANKEM). All the amino acids such as Cit, Val, Phe, Lys, Trp, Asp are naturally occurring amino acids with S-configuration. In several examples, tetrapeptide and unnatural amino acids can also be used. Flash chromatography was performed on 230-400 mesh silica gel with the indicated solvent systems. Proton Nuclear magnetic resonance spectra were recorded on Bruker Spectrometer at 400MHZ using DMSO as solvent.
  • Peak positions are given in parts per million downfield from tetramethylsilane as the internal standard. J values are expressed in hertz. Mass analyses were performed on (Agilent/Shimadzu) spectrometer using electrospray (ES) technique. HPLC analyses were performed on (Agilent/Waters), PDA-UV detector equipped with a Gemini C-18 (1000x 4.6 mm; 5u) and all compounds tested were determined to be >95% pure using this method. As can be seen in many protease-cleavable linker-payload conjugates, two peaks were isolated at the end of the reaction. The Peak-A (or Peak-1) is the desired compound with the stereochemistry as shown.
  • Exatecan-linker conjugate precursors suitable for preparing an NDC of the present disclosure can be synthesized according to the following protocols.
  • the exatecan-linker conjugate precursors comprise a terminal azide group, they are suitable for attaching to a nanoparticle functionalized with alkyne moieties (e.g., DBCO), using click chemistry.
  • alkyne moieties e.g., DBCO
  • reaction mixture was quenched with water (15 mL) and extracted with 10% methanol in chloroform (2x20 mL). The combined organic layers were dried over anhydrous sodium sulfate (Na 2 SO 4 ) and concentrated under reduced pressure.
  • Folic acid conjugate precursors suitable for preparing a folate receptor targeting NDC disclosed herein can be prepared according to one of the following synthetic protocols.
  • the folic acid conjugate precursors comprise a terminal azide group, they are suitable for attaching to a nanoparticle functionalized with alkyne moieties (e.g., DBCO), using click chemistry.
  • alkyne moieties e.g., DBCO
  • reaction mixture was extracted with DCM and water, and the organic layer was dried over Na 2 SO 4 , and evaporated under vacuum. The residue was purified by flash chromatography and dried under vacuum to provide tert-butyl (1-azido-30-oxo-3,6,9,12,15,18,21,24,27-nonaoxa-31-azatritriacontan-33-yl)carbamate ( 465 ; 0.45 g) as a liquid.
  • Aqueous synthesis methodology can be used for the preparation and functionalization of ultrasmall nanoparticles of the present disclosure.
  • methodology based on the procedures outlined in WO 2016/179260 A1 and WO 2018/213851 A1 (the contents of which are incorporated herein by reference in their entireties) may be used.
  • a fluorescent compound such as, but not limited to Cy5
  • a fluorescent compound can be functionalized with a maleimide group, to provide a maleimide-functionalized fluorescent compound that has a net positive charge.
  • This can be conjugated with a thiol-silane, such as (3-mercaptopropyl)trimethoxysilane (MPTMS) to produce a silane-functionalized fluorescent compound such as Cy5-silane.
  • MTMS (3-mercaptopropyl)trimethoxysilane
  • the conjugation may be performed in dimethyl sulfoxide (DMSO) in a glovebox under inert atmosphere overnight (16-24 hours) and at room temperature (18-25 °C).
  • DMSO dimethyl sulfoxide
  • the next step of the synthesis can be performed in a suitable chamber, such as a glass flask, container, or reactor, and can involve stirring deionized water with a pH of around 8.5-10.5 which can be achieved using an aqueous solution of ammonium hydroxide of pH 7.5-8.5.
  • a silica precursor such as a tetraalkyl orthosilicate, e.g., tetramethyl orthosilicate (TMOS)
  • TMOS tetramethyl orthosilicate
  • the reaction can be left stirring at room temperature overnight (1-48 hours), to provide silica cores encapsulating the fluorescent compound, e.g., Cy5 dye.
  • a PEG-silane can be added into the reaction under stirring at room temperature to coat the silica core with PEG molecules, and the reaction can be left stirring for 1-48 hours. This step may be followed by heating between 75-85 °C for 1-48 hours. The reaction can then be cooled down to room temperature and purified (e.g., including sterile filtration to remove aggregates formed as side-product of the reaction, and bacteria if any present). Further functionalization of the nanoparticle may then be performed.
  • purified e.g., including sterile filtration to remove aggregates formed as side-product of the reaction, and bacteria if any present.
  • a nanoparticle prepared using a method disclosed herein may be further functionalized, e.g., using a method outlined in FIGS. 2 or 3 , or in Scheme 6 below.
  • a method outlined in FIGS. 2 or 3 or in Scheme 6 below.
  • 3-cyclopentadienylpropyl)triethoxysilane (diene-silane) can be used to functionalize a nanoparticle (e.g., C'Dot) with cyclopentadiene groups, then DBCO-PEG-maleimide can be reacted with the diene-functionalized nanoparticle to provide a DBCO-functionalized nanoparticle.
  • Cy5-C'Dot (which may be prepared using a method described herein) was diluted with deionized water to a desired concentration, typically between 15 to 30 ⁇ M, in a round-bottom flask with a stir bar.
  • a desired concentration typically between 15 to 30 ⁇ M
  • 3-Cyclopentadienylpropyl)triethoxysilane (cyclopentadiene) was first diluted 100x in DMSO and then added into the reaction with stirring, to reach a desired particle to cyclopentadiene molar ratio. After overnight reaction, 10x PBS was added into the reaction to achieve a final concentration of 1x PBS.
  • a DBCO-maleimide precursor e.g., DBCO-PEG4-maleimide
  • DMSO dimethyl methacrylate
  • DBCO-PEG4-maleimide a DBCO-maleimide precursor
  • DMSO dimethyl methacrylate
  • the reaction solution was then concentrated and purified using gel permeation chromatography (GPC) to obtain diene-based DBCO-C'Dot.
  • the purification may be performed based on the principle of size separation. Aggregates and free small molecules having molecular weight different than that of the pegylated nanoparticles are separated using gel permeation chromatography columns (GPC) or Tangential Flow Filtration (TFF) system. Two different membranes, 300 kDa, and 50 kDa cut-off sizes were employed for the removal of large aggregates and free small molecules respectively. Both GPC and TFF systems can be used to transfer the aqueous medium to water, saline etc. Purified DBCO-C'Dot in deionized water can be sterile filtered again and the quality control (QC) steps can be performed, followed by storage in refrigerator at 2-8 °C.
  • GPC gel permeation chromatography columns
  • TFF Tangential Flow Filtration
  • the neutral charge of the cyclopentadiene groups averts hydrolysis of the amide bonds in the linkage, that can be accelerated by other types of precursors (e.g., when using amine-silanes instead of diene-silanes, the primary amine groups can cause hydrolysis).
  • the NDCs produced using this method are highly stable (see, e.g., comparison in FIGS. 33A-33B ).
  • diene-functionalized nanoparticles e.g., cyclopentadiene-functionalized nanoparticles
  • NDCS nethyl-sethyl-sethyl-sethyl-sethyl-sethyl-sethyl-sethyl-sethyl-sethyl-sethyl-sethyl-sethyl-s
  • NDCs of the present disclosure comprising the nanoparticle (also referred as C'Dot), targeting ligand (folic acid) and linker-drug conjugates can be prepared as outlined in the flow chart presented in FIG. 3 , and in Scheme 7 below.
  • a desired number of targeting ligands per nanoparticle can be achieved.
  • nanoparticles of the present disclosure may be functionalized to contain about 10 to about 20 folic acid moieties, e.g., about 10, about 11, about 12, about 13, about 14, or about 15 folic acid moieties.
  • nanoparticles of the present disclosure may be functionalized to contain about 10 to about 40 exatecan-linker moieties, e.g., about 20, about 21, about 22, about 23, about 24, or about 25 exatecan moieties.
  • DBCO-C'Dot (referred as C'Dot in FIG. 3 ) was diluted using deionized water to a concentration of 15-45 ⁇ M. After the temperature of DBCO-C'Dot solution was around 18-25 °C, folate receptor (FR)-targeting ligand precursor such as, folic acid (FA) functionalized with an azide (compound 606 prepared in Example 2) was dissolved in DMSO (0.021 M) and was then added into the reaction with stirring at room temperature, providing a C'Dot functionalized with FA via the DBCO group on the surface.
  • FR folate receptor
  • FA folic acid
  • the reaction ratio between DBCO-C'Dot and FA was kept from 1:5 to 1:30, and the solution was stirred for 16-24 hours at temperature of 18-25 °C.
  • FR-targeting ligand addition is followed by sterile filtration, purification and QC testing to yield FA-C'Dot (referred as C'Dot intermediate in FIG. 3 ), and can be stored in a refrigerator at 2-8 °C.
  • FA-C'Dot comprises a portion of DBCO groups that are available for further click-reactions, e.g., with molecules with azide functionality.
  • the folate-targeting ligand e.g., folic acid
  • the volume of the FR-targeting ligand conjugation reaction can range from 5 mL to 30 L, and the concentration of DBCO-C'Dot can range from 15 to 45 ⁇ M.
  • concentration of DBCO-C'Dot can range from 15 to 45 ⁇ M.
  • the following parameters are given for a typical reaction volume of 600 mL and a DBCO-C'Dot concentration of 25 ⁇ M.
  • the ratio of DBCO-C'Dot to FR-targeting ligands was precisely controlled to obtain the desired number of FR-targeting ligands per particle, and typically can range from 1:5 to 1:30.
  • folate-PEG-azide was dissolved in DMSO to a concentration of 0.021 M, and 8.571 mL of the folate-PEG-azide/DMSO solution was added into the reaction. After stirring overnight at room temperature, the reaction mixture was either purified to obtain FA-C'Dot or continue directly to next conjugation step if the purity of FA-C'Dot is no less than 95%.
  • the conversion rate of FR-targeting ligand is typically higher than 95%.
  • the number of folic acid groups attached onto each FA-C'Dot was characterized by UV-Vis, and a representative UV-Vis absorbance spectrum is shown in FIG. 4 .
  • the number of DBCO groups on each C'Dot can be calculated using the extinction coefficient of C'Dot and DBCO groups
  • FA-targeted NDC or FA-CDC
  • FA-C'Dots were diluted using deionized water to a concentration of 15-45 ⁇ M.
  • exatecan-linker conjugate precursor e.g., compound 202 described in Example 1
  • cathepsin dissolved in DMSO 0.04 M
  • This step functionalized the FA-C'Dot with the linker-drug conjugate via the available DBCO groups on the surface.
  • the reaction ratio between FA-C'Dot and linker-drug conjugate was kept around 1:10-1:50 and the solution was stirred for 16-24 hours.
  • the addition of linker-drug conjugate was followed by sterile filtration, and purification.
  • FA-CDC also referred as NDC
  • the volume of the cleavable exatecan conjugation reaction can range from 5 mL to 30 L, and the concentration of FA-C'Dot can range from 15 to 45 ⁇ M.
  • the following parameters are given for a typical reaction volume of 600 mL and a FA-C'Dot concentration of 25 ⁇ M.
  • the ratio of FA-C'Dot to cleavable exatecan was precisely controlled to obtain the desired number of cleavable exatecan per particle, and typically can range from 1:10 to 1:60.
  • cleavable exatecan was dissolved in DMSO to a concentration of 0.04 M, and 15 mL of the cleavable exatecan/DMSO solution was added into the reaction. After stirring overnight at room temperature, the reaction mixture was purified to obtain FA-CDC.
  • the number of exatecan payloads attached onto each NDC may be characterized by UV-Vis.
  • a representative UV-Vis absorbance spectrum is shown in FIG. 5 .
  • the number of exatecan payloads on each C'Dot can be calculated using the extinction coefficient of C'Dot and Exatecan at 360 nm after the subtraction of the absorption of Folic Acid at the same wavelength.
  • a nanoparticle may be functionalized with a folate receptor targeting ligand and a payload-linker conjugate in any order (e.g., the protocol outlined above for functionalizing the nanoparticle with exatecan may be carried out prior to the protocol for conjugating folic acid).
  • the average diameter of NDCs can be measured by any suitable methods, such as, but not limited to fluorescence correlation spectroscopy (FCS) ( FIG. 6 ) and gel permeation chromatography (GPC) ( FIG. 7 ).
  • FCS fluorescence correlation spectroscopy
  • GPS gel permeation chromatography
  • FCS detects the fluorescence fluctuation resulted from particle diffusion through the focal spot on the objective. Particle diffusion information is then extracted from the autocorrelation of signal intensity fluctuations, from which the average hydrodynamic particle size can be obtained by fitting the autocorrelation curve using a single-modal FCS correlation function.
  • the average hydrodynamic diameter of NDC was about 6 nm to about 7 nm ( FIG. 6 ).
  • GPC is a type of molecular sieving chromatography, where the separation mechanism is based on the size of the analyte (here NDC's).
  • NDC's analyte
  • the elution time of NDC is compared to a series of proteins with varying molecular weight. The results suggest that the elution time of NDC's is comparable to that of protein standards with molecular weight between 158 kDa and 44 kDa, consistent with the particle size average hydrodynamic size of about 6.4 nm ( FIG. 7 ).
  • RP-HPLC reverse phase HPLC
  • RP-HPLC reverse phase HPLC
  • RP-HPLC reverse phase HPLC
  • the nanoparticles are well separated from aggregates and other chemical moieties such as targeting ligands that are non-covalently associated with the nanoparticles and degraded products. Different chemical moieties are identified based on their elution time and unique UV/Vis spectra.
  • the photodiode array detector collects UV-Vis spectra from 210 to 800 nm, and impurities of interest are measured at 330 nm.
  • a representative chromatogram shown for the NDCs in FIG. 8 suggests that the purity of NDCs of the present disclosure is higher than 99.0 %.
  • NDCs of the present disclosure comprise a linker-payload conjugate, e.g., a protease-cleavable linker, such as cathepsin-B (Cat-B) cleavable linker.
  • a linker-payload conjugate e.g., a protease-cleavable linker, such as cathepsin-B (Cat-B) cleavable linker.
  • the NDCs may release the payload (i.e., exatecan).
  • the drug-releasing profile and the stability of linker-drug conjugates on the nanoparticle were tested according to the following protocols.
  • Exatecan exhibits an absorption maximum at a wavelength of around 360 nm ( FIG. 9 ), and this signal can be used to trace the payloads in high-performance liquid chromatography (HPLC) for releasing and stability studies.
  • HPLC high-performance liquid chromatography
  • the amount of released drugs vs non-released drugs was measured using reverse phase HPLC by analyzing the area under curve (AUC) ( FIG. 10A and FIG. 10B ).
  • the seal wash used for the system was composed of 90% 18.2 M ⁇ •cm deionized water and 10% methanol (HPLC grade, VWR).
  • the injection needle was washed using a mixture of 25 vol% 18.2 M ⁇ •cm deionized water, 25 vol% acetonitrile, 25 vol% methanol, and 25 vol% 2-propanol.
  • Samples were prepared in a concentration range of 0.25 to 2 ⁇ M and the injection volume ranges from 60 ⁇ L to 10 ⁇ L, respectively. Higher sample concentration can be used if detector signal is low.
  • Vials used for all injections are fresh Waters Total Recovery vials with screw caps that have pre-slit PTFE septa (part number 186000385C).
  • the PDA lamp was turned on and allowed to warm up for at least 30 minutes.
  • the system and column were equilibrated with 95% 0.01% TFA in deionized water, 5% acetonitrile for at least 10 minutes at a flow rate of 1.0 mL/min after the PDA lamp had warmed up.
  • the gradient used began at 95% 0.01% TFA in deionized water and 5% acetonitrile and linearly changed to 15% 0.01% TFA in deionized water, 85% acetonitrile over 8 minutes.
  • Acetonitrile composition was increased to 95% over an additional minute and held at 95% for an additional 2 minutes to ensure that any strongly retained compounds are eluted.
  • the composition of the solvent was then changed back to the starting composition of the gradient over an additional minute and allowed to equilibrate for 3 minutes before another injection began. Between sample injections a blank injection was run to ensure that no carryover occurred.
  • Cat-B cathepsin B
  • 2 ⁇ L, 0.33 ⁇ g/ ⁇ L of Cat-B was first added with 300 ⁇ L of activation buffer (25 mM MES, 5 mM DTT, pH 5.0), forming 2.2 ⁇ g/mL of Cat-B.
  • activation buffer 25 mM MES, 5 mM DTT, pH 5.0
  • the mixture was kept at room temperature for 15 min before use.
  • 100 ⁇ L of 2 ⁇ M drug-nanoparticle-conjugate was mixed with 100 ⁇ L of activated Cat-B. The mixture was then transferred to 37 °C.
  • FIGS. 11A-11C The RP-HPLC chromatograph of three representative NDCs (NDC B, NDC C, and NDC D) at different time points after incubation with cathepsin-B is depicted in FIGS. 11A-11C , respectively.
  • FIG. 12A depicts the T 1/2 as 2.9 hours for NDC B.
  • FIG. 12B depicts the T 1/2 as 2.6 hours for NDC C.
  • FIG. 12C depicts the T 1/2 as 1.4 hours for NDC D.
  • NDC Stability Test To assess the drug releasing profile and stability of the linker-drug conjugates under non-cleavage conditions, an exemplary NDC was incubated in phosphate-buffered saline (PBS) buffer or animal serum at 37 °C. The NDC was prepared according to Example 3, using the exatecan-linker conjugate precursor 202 from Example 1)
  • the linker-payload conjugate of the NDC (as prepared in Example 3, using the exatecan-linker conjugate precursor 202 from Example 1) is stable, as 5% of less of the exatecan was released from the linker drug conjugate after 24 hours under non-cleavage conditions, i.e ., when maintained in PBS, human serum, or mouse serum.
  • NDCs disclosed herein were tested according to the following protocols. NDCs used were prepared according to Example 3, using the exatecan-linker conjugate precursor 202 of Example 1. The amount of folic acid per nanoparticle, and the amount of exatecan per nanoparticle, could be adjusted according to the protocol outlined in Example 3.
  • Human KB cell line, SKOV-3 cells and TOV-1 12 cell line were purchased from ATCC.
  • I-GROV1 human ovarian carcinoma cell line was purchased from EMD Millipore.
  • Cells were maintained in Folic Acid free RPMI 1640 media/10% FBS, and 1% of penicillin/streptomycin, unless otherwise specified.
  • Cancer cells were cultured in folic acid-free medium (RPMI1640, ThermoFisher, GIBCO) for at least one week before the study.
  • the competitive binding study ( FIG. 13 ) was performed using the NDC of Example 3.
  • the active targeting of NDC can be fully blocked by incubating with the presence of 1 mM free Folic Acid.
  • the competitive binding study shows >40-fold enhancement in binding capability of the NDC when compared with free folic acid, demonstrating the presence of a multivalent effect when conjugating multiple folic acid ligands on each ultrasmall C'Dot ( FIG. 13 ).
  • the folate receptor targeting can be blocked by competitive binding of free folic acid, such as by incubating with the presence of 1 mM free Folic Acid.
  • the flow cytometry shows comparable folate receptor targeting efficacy of two NDC formulations with varied folic acid ligand density, in KB cell line.
  • the linker-exatecan conjugate precursor used to prepare the NDCs in this study is described in Example 1 (Compound 202).
  • the blocking group has 1 mM of free Folic Acid. ( FIG. 14 ).
  • the flow cytometry shows comparable folate receptor targeting efficacy of three NDCs in KB cell line with varied drug per particle (DPR) (i.e., number of exatecan molecules per nanoparticle).
  • the blocking group involved blocking receptors with 1 mM of free folic acid.
  • the NDCs with different ratios of exatecan per nanoparticle were prepared using Compound 202 described in Example 1, and the results of the study are provided in FIG. 15 . All FA-CDCs comprise between 12 and 22 folic acid moieties.
  • FA-CDCs with high drug-particle ratio (DPR) comprise between 35 and 50 exatecan-linker conjugate groups.
  • FA-CDCs with medium DPR comprise between 17 and 25 exatecan-linker conjugate groups.
  • FA-CDCs with low DPR have between 5 and 10 exatecan-linker conjugate groups.
  • the in vitro cytotoxicity of the NDCs disclosed herein were tested in cancer cells.
  • the cancer cells were cultured in folic acid-free medium (RPMI1640, ThermoFisher, GIBCO) for at least one week before the study.
  • Cells were plated in opaque 96-well plates at a density of 3 ⁇ 10 3 cells per well (total of 90 mL) and allowed to attach overnight.
  • the following day, cells were treated with NDC (prepared according to Example 3) at a concentration range of 0-50 nM (0, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50 nM) by adding 10 mL of 10x stock FA-CDC solution.
  • Cells were treated for a pre-defined exposure time (depending on the study design, e.g., 4-6 hours, or 7 days).
  • a pre-defined exposure time e.g., 4-6 hours, or 7 days.
  • cancer cells in each wells were washed with 100 mL PBS and refreshed with 100 mL of cell medium. After washing, the plates were returned back to 37 °C incubator for 7 days before the viability assay.
  • 7-day-exposure-time viability study no additional washing step was performed. After 7 days, the cell viability was assessed using the CellTiter-Glo2.0 assay (Promega) according to manufacturer's instructions. Data for both viability and proliferation were plotted using Prism7 software (GraphPad).
  • a 2D confocal imaging study was carried out to determine the targeting of cells with varying levels of folate-receptor availability, using two exemplary NDCs.
  • the cells with high folate-receptor expression (denoted ++++) were KB cells.
  • the cells with no FR expression (denoted (-) were TOV-112D cell line. FR-blocked cells were also used.
  • KB cells were maintained in folic acid free RPMI 1640 media with 10% FBS, 1% penicillin/streptomycin.
  • TOV-112D cells were maintained in 1:1 mixture of MCDB 105 medium containing a final concentration of 1.5 g/L sodium bicarbonate and Medium 199 containing a final concentration of 2.2 g/L sodium bicarbonate, supplemented with 15% FBS and 1% penicillin/streptomycin.
  • Cells were trypsinized and seeded in 8-well Lab-Tek chambered coverglass, at 1.0 ⁇ 105 cells per well, and cultured overnight to allow for attachment.
  • NDCs were prepared according to Example 3 and are displayed below in Table 4.
  • NDC D was prepared using the linker-payload conjugate ( 202 ) described in Example 1. Table 4. Exemplary NDCs used in 2D Confocal Imaging Assay.
  • L7526 ex/em504/511 nm was added to final concentration of 100 nM in folic acid free RPMI 1640 media with 10% FBS, 1% P/S, and incubated at 37 °C for 45 min. Cells were washed once to remove remaining lysotracker dyes. To stain nuclei, Hoechst 33342 solution (Thermo Fisher Cat.62249, 20 mM) was diluted 1:4000 in Folic Acid free RPMI 1640 media with 10% FBS, 1% P/S, and incubated at 37 °C for 10 min.
  • Cells were washed once, and media was exchanged to phenol red free RPMI 1640 media for confocal imaging using Nikon spinning disk confocal microscope, 60x objective, 405 nm, 488 nm, 640 nm laser lines, exposure time 100 ms for 405 channel, 500 ms for 488 channel, and 600 ms for 640 channel.
  • results of confocal microscopy of NDC B are provided in FIG. 17
  • results for NDC D are provided in FIG. 32 .
  • These images demonstrated the highly specific active targeting and lysosome trafficking of the NDCs of the present disclosure, indicating that once the FA-targeting NDCs bind to cells they become internalized in folate receptor positive cell lines, where the exatecan payload may be cleaved (e.g., by cathepsin-B) to release free exatecan in the cancerous cell.
  • a 3D tumor spheroid model assay was conducted to determine the tumor penetration of the NDCs disclosed herein.
  • the assay compared an exemplary NDC (prepared according to Example 3, using exatecan-linker conjugate precursor 202 of Example 1), with a payload-free FA-targeting nanoparticle (also prepared according to Example 3, with only the FA precursor and without exatecan-payload conjugate precursor); a folate receptor (FR)-targeting ADC; and the corresponding payload-free FR-targeting antibody.
  • the FR-targeting antibody was prepared based upon the published sequence of mirvetuximab (provided in U.S. Patent No. 9,637,547 as huMov19; the contents of which are incorporated herein by reference in its entirety).
  • the ADC was prepared with the same antibody and was conjugated to the maytansinoid drug DM4 (created by Syngene International Ltd.) via a 4-(pyridin-2-yldisulfanyl)-2-sulfo-butyric acid (sSPDB) linker (based on the linker used in U.S. Patent No. 9,637,547 ).
  • the ADC and antibody were each conjugated with Cy5 organic dye, by reaction with Cy5-NHS ester, and were purified by a PD-10 column.
  • Corning ultra-low attachment surface 96-well spheroid microplates were utilized in seeding KB cells for having KB spheroids with cell density 10,000 / well.
  • Single-cell suspensions were generated from trypsinized monolayers and diluted to 100,000 cells/mL using RPMI medium (folic acid free). 100 mL of cell suspension were dispensed into each well of a microplate. The plate was kept in an incubator for 24 hours for cells forming spheroids. KB cell spheroids can be easily observed by microscope with 10X objective.
  • NDC prepared according to Example 3, using exatecan-linker conjugate precursor 202 of Example 1
  • F-C'Dot folate-targeted nanoparticles
  • FR-targeted ADC FR-targeted ADC
  • results from the Z-stack confocal microscope imaging of KB tumor spheroid treated with the NDC, FA-C'Dot, FR-targeted ADC, and payload-free FR-targeted antibody is depicted in FIG. 18 .
  • the results show that the penetration and well diffusion of NDC and FA-C'Dots throughout the whole >800 mm of tumor spheroids.
  • labeled antibody and ADC merely accumulated around, but not inside of, the tumor spheroids.
  • the ability of the NDCs disclosed herein to achieve efficient tumor penetration is highly advantageous, and shows significant improvement compared to conventional drug delivery platforms.
  • a radiolabeling assay was conducted to determine the in vivo biodistribution of the folate receptor-targeting NDCs of the present disclosure.
  • the NDCs used for the assay were conjugated with the chelator desferrioxamine (DFO) and then bound with a radionuclide ( 89 Zr).
  • DFO desferrioxamine
  • PET/CT imaging data were normalized to correct for nonuniformity of response, dead-time count losses, positron branching ratio, and physical decay to the time of injection; no attenuation, scatter, or partial-volume averaging corrections were applied.
  • the counting rates in the reconstructed images were converted to activity concentrations (percentage injected dose per gram of tissue, % ID/g) by use of a system calibration factor derived from the imaging of a mouse-sized water-equivalent phantom containing 89 Zr.
  • Region-of-interest (ROI) analyses of the PET data were performed using IRW software.
  • organs from each individual mouse were collected, wet-weighted and gamma counted (Automatic Wizard2 ⁇ -Counter, PerkinElmer).
  • the uptake of 89 Zr-DFO-FA-CDC was presented as % ID/g (mean ⁇ SD).
  • the NDCs of the present disclosure enable precise tumor targeting, deep tumor penetration and high tumor killing efficacy.
  • the NDCs can be cleared rapidly and efficiently from the body, which reduces the potential for off-target toxicities and results in an improved safety profile.
  • the NDCs disclosed herein (comprising targeting ligands (folic acid) and payload (exatecan)) can be administered to a subject and circulate through the blood stream, target the cancer (e.g., tumor), diffuse, penetrate, internalize, and cleave the exatecan payload, killing the cancer cells.
  • the renal clearance and biodistribution pattern of FA-CDC were tested.
  • FIG. 19A after the intravenous injection, the 89 Zr-DFO-FA-CDC circulated in the blood stream of healthy nude mouse, as indicated by the high radioactive signal from the heart and artery. Dominant radioactive signal can also be seen from the mouse bladder, demonstrate the renal clearance of the NDC.
  • the majority of the injected 89 Zr-DFO-FA-CDC was cleared out of the mouse body.
  • the changes in biodistribution pattern at 2 hours and 24 hours post-injection is also shown in FIG. 19B .
  • the NDC can circulate in the blood stream with a dominant renal clearance pathway, whilst avoiding clearance by the mononuclear phagocytic system (MPS) (i.e., liver and spleen).
  • MPS mononuclear phagocytic system
  • the in vivo efficacy of the NDC was carried out using a human KB tumor mouse model.
  • the assay compared the NDC prepared according to Example 3 using the exatecan-payload conjugate precursor 202 (Example 1; here labeled D, and shown in FIG. 20D , with the NDCs indicated in Table 5 below (NDCs A-C and E-F).
  • NDCs A-C and E-F Each NDC was compared to a control and free exatecan, and NDCs E and F were compared to free exatecan and irinotecan (CPT-11). Table 5.
  • Each payload-linker is conjugated to the NDC via a DBCO moiety (prepared according to the protocol outlined in Example 3).
  • Human KB cell line was purchased from ATCC and maintained in folic acid free RPMI 1640 media/10% FBS, and 1% of penicillin/streptomycin, unless otherwise specified. Once the KB cells were cultured to reach an adequate cell count, the cell viability was confirmed by a hemocytometer and trypan blue staining assay.
  • each mouse was injected with KB cells at a density of 2x106 cells/mice at 0.1 mL Matrigel/cell dilution volume per injection on the left lower flank of the thigh. Once a subcutaneous tumor volume has reached a palpable size of 75 to 150 mm 3 in a required number of mice for this study, the mice was randomized and assigned to each treatment cohort resulting with comparable tumor volume statistics. Following randomization and study cohort assignment, each dose cohort was treated according to the routes of administration, dosage and schedule.
  • NDCs B-D Two dose levels of each of NDCs B-D were used in the efficacy study (only one dose level for NDCs A, E and F).
  • Tumor volume measurements were performed using a calibrated caliper every second day during the dose treatment period, followed by twice weekly measurements during the recovery period of the in-life phase, and tumor volumes were determined using the formula length (mm) ⁇ width (mm) ⁇ width (mm) ⁇ 0.50.
  • Body weight measurements were performed every second day during the dose treatment period, followed by twice weekly measurements during the recovery period of the in-life phase. Mice were euthanized when the end points of the study reached 1000 mm 3 . Tumors were harvested and tumor size was measured. Tumor were surgically excised and snap-frozen for storage at -80 °C until future analysis.
  • the tumor growth charts depicted for the in vivo efficacy study shows a clear response of tumor growth inhibition in mice treated with the NDC prepared according to Example 3 using the exatecan-payload conjugate precursor 202 (from Example 1), which is shown in FIG. 20D .
  • growth inhibition was observed in NDC A ( FIG. 20A ), NDC B ( FIG. 20B ), and NDC C ( FIG. 20C ).
  • mice treated with NDC E ( FIG. 20E ), and NDC F ( FIG. 20F ) showed no significant inhibition in tumor growth.
  • Doses for the NDCs are provided in FIGS. 20A - 20F . Clear response of tumor growth inhibition was observed in mice treated with NDCs A-D. Control group mice received normal saline follow the same Q3DX3 dose regimen.
  • NDCs disclosed herein were prepared according to Example 3, using the exatecan-linker conjugate precursor 202 (from Example 1).
  • Naive human KB cell line were purchased from ATCC and maintained in folic acid free RPMI 1640 media/10% FBS, and 1% of penicillin/streptomycin.
  • the cells in flask 50-60% confluence were repeatedly treated with increasing concentration of exatecan, topotecan, SN-38 or irinotecan for over 4 months.
  • the starting TOP1 inhibitor treatment concentration was close to the KB cell's IC 90 values.
  • the cells were carefully washed with fresh RPMI 1640 media and left to proliferate for an additional 2-3 days until reaching 50-60% confluence.
  • the next round of TOP1 inhibitor treatment was started with 2-10X higher TOP1 inhibitor concentration.
  • Resistant factor and IC 50 assay are identical to Resistant factor and IC 50 assay.
  • FIG. 21A shows the IC 50 curves of irinotecan in both naive and resistant KB cells, which demonstrates the successful development of 5X irinotecan-resistant KB cells, where IC 50 free irinotecan in irinotecan-resistant KB cells was 3,618 nM, compared to 668 nM in naive cells.
  • FIG. 21A shows the IC 50 curves of irinotecan in both naive and resistant KB cells, which demonstrates the successful development of 5X irinotecan-resistant KB cells, where IC 50 free irinotecan in irinotecan-resistant KB cells was 3,618 nM, compared to 668 nM in naive cells.
  • NDC NDC
  • FIG. 22A shows the IC 50 curves of exatecan in both naive and resistant KB cells, which demonstrates the successful development of >8X exatecan-resistant KB cells, where IC 50 of exatecan in regular KB cells was 2 nM, compared with 4 nM in KB cells pretreated 4x with exatecan, and 16.9 nM in KB cells pretreated 7x with exatecan.
  • FIG. 22A shows the IC 50 curves of exatecan in both naive and resistant KB cells, which demonstrates the successful development of >8X exatecan-resistant KB cells, where IC 50 of exatecan in regular KB cells was 2 nM, compared with 4 nM in KB cells pretreated 4x with exatecan, and 16.9 nM in KB cells pretreated 7x with exatecan.
  • FIG. 22A shows the IC 50 curves of exatecan in both naive and resistant KB cells, which demonstrates the successful development of >8X exatecan-resistant
  • FIG. 22B shows the IC 50 curves of the NDC (FA-CDC) (prepared according to Example 3, using the exatecan-linker conjugate precursor 202 of Example 1) in both naive and resistant KB cells (4x or 7x pretreatment), where the IC 50 of the FA-CDC was 0.27 nM, 0.28 nM, and 0.30 nM, respectively.
  • Example 12 Activity of NDCs in cancer cells with varied folate receptor expression levels
  • NDCs exemplary NDCs
  • the NDCs were prepared according to Example 3, using the payload-linker conjugate precursor 202, of Example 1.
  • the NDCs (FA-CDCs) tested had a drug-to-particle ratio (DPR) of 43, 20, 8, and 1 (i.e., 43, 20, 8, and 1 exatecan-linker groups per nanoparticle).
  • DPR drug-to-particle ratio
  • Cancer cells with varied FR alpha expression levels (KB (++++), IGROV-1(++), SK-OV-3(++), HCC827(++), A549(-), and BT549(-)) were cultured in folic acid-free medium (RPMI1640, ThermoFisher, GIBCO) for at least one week before the study. Assays for 7-day exposure and 6-hour exposure were both conducted.
  • Cells were plated in opaque 96-well plates at a density of 3x103 cells per well (total of 90 ⁇ L) and allowed to attach overnight. The following day, cells were treated with NDC with varied drug-to-particle ratio (DPR) at a concentration ranging from 0-50 nM (0, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50 nM) by adding 10 ⁇ L of 10x stock compounds.
  • DPR drug-to-particle ratio
  • NDC non-small cell lung cancer
  • TNBC triple negative breast cancer
  • H&N head and neck cancer
  • the cytotoxic efficacy was determined by KIYATEC using the KIYA-PREDICT TM assay.
  • the FR ⁇ immunohistochemistry (ICH) scoring of tumor tissue from platinum-resistant ovarian, endometrium, non-small cell lung, breast, triple-negative breast, head & neck cancer patients were conducted by XenoSTART by using the Biocare Medical FRa IHC Assay Kit (cat # BRI4006KAA), following the manufacturer's protocol.
  • a total of 28 PDX models from different indications were selected based on the IHC scores and provided to KIYATEC for the KIYA-PREDICTTM assay.
  • cryopreserved PDX tumors were thawed and enzymatically dissociated to single cells, and plated into 384-well spheroid microplates (Corning).
  • Flow cytometry was also performed to assess the FR ⁇ levels among different PDX models. Following the 24 hours of spheroid formation, NDC or controls were added at the designed concentration range and incubated for 7 days. After that, the cell viability was measured by CellTiter-Glo ® 3D (Promega). The data was analyzed in Microsoft Excel and GraphPad Prism.
  • Example 14 In vitro and in vivo efficacy of an exemplary NDC in pediatric acute myeloid leukemia models
  • Cancer cells (IGROV-1 and AML MV4;1 1 cell lines) were cultured in folic acid-free medium (RPMI1640, ThermoFisher, GIBCO) for at least one week before the study.
  • PBS cold phosphate-buffered saline
  • BSA bovine serum albumin
  • PE anti-FR alpha phycoerythrin
  • PE anti-FR alpha antibody-PE
  • a non-targeted CDC and isotype antibody-PE were used as negative controls for the exemplary NDC and anti-FR alpha antibody-PE, respectively.
  • the cell suspension was then stained with viability kit (LIVE/DEAD TM Fixable Violet Dead Cell Stain Kit, Thermo Fisher) for 10-15 min.
  • the cells were next centrifuged (2000 revolutions per minute, 5 min), washed (2-3 times) using cold PBS (with 1% of BSA) before resuspending in PBS (with 1% of BSA).
  • Triplicate samples were analyzed on a LSRFortessa flow cytometer (BD Biosciences) (Cy5 channel, 633 nm/647 nm, Live/dead cell stain, 405 nm). Results were processed using FlowJo and Prism 7 software (GraphPad).
  • FIGS. 25A-25D The flow cytometry histograms of the exemplary NDC and anti-FR alpha antibody-PE compared with the respective negative controls (non-targeted NDC or isotype antibody-PE) are shown in FIGS. 25A-25D .
  • the flow study demonstrates the specific FR alpha targeting capability of the exemplary NDC to both the IGROV-1 (FR alpha positive human ovarian cancer) and the AML MV4;11 cell lines.
  • Cancer cells (IGROV-1 and AML MV4;1 1 cell lines) were cultured in folic acid-free medium (RPMI1640, ThermoFisher, GIBCO) for at least one week before the study. Cells were plated in opaque 96-well plates at a density of 3 ⁇ 10 3 cells per well (total of 90 ⁇ L) and allowed to attach overnight. The following day, cells were treated with the exemplary NDC at a concentration ranging from 0-100 nM, by adding 10 ⁇ L of 10x stock NDC solution. For the shorter exposure viability study, cells were treated for 4 hours and washed (3x) with 100 ⁇ L PBS.
  • folic acid-free medium RPMI1640, ThermoFisher, GIBCO
  • FIGS. 26A-26B The in vitro specific cytotoxic activity of the exemplary NDC in FR alpha positive human ovarian cancer and MV4; 11 AML cell lines is displayed in FIGS. 26A-26B .
  • Cells were treated with the exemplary NDC at the indicated concentrations, incubated at 37 °C for 4 hours, washed, and returned to the incubator for an additional 5 days, before performing the CellTiter-Glo ® cytotoxic assay.
  • NDC cell line-derived xenograft
  • mice burden and response to treatments was monitored using non-invasive bioluminescent imaging (from both the front and the back of the mouse), and flow cytometry analysis of mouse peripheral blood drawn by submandibular bleeds was carried out bi-weekly, starting from the first week of CDC treatment. Mice were monitored for disease symptoms (including tachypnea, hunchback, persistent weight loss, fatigue, and hind-limb paralysis). Mice from the saline control group (Cohort 1) were euthanized due to the high AML burden on Day 44 post-leukemia injection (tissues including blood, bone marrow, thymus, liver, lungs and spleen were harvested at necropsy and analyzed for the presence of leukemia cells). Mice from the treatment groups (Cohorts 2-4) continued to receive weekly bioluminescent imaging and bodyweight monitoring. An illustration of the timeline for mice preparation, treatment, and imaging is provided in FIG. 31 .
  • mice All the mice were randomized prior to dosing and weighed to provide the correct designed dose based on Table 6 below. Leukemia burden and response to treatments was monitored weekly using non-invasive bioluminescent imaging. Bodyweight was measured every other day. The mice were terminated if their weight loss was over 20%. Table 6.
  • FIG. 27 provides the bodyweight change of AML mice treated with normal saline and the exemplary NDC at the three dose levels indicated in Table 6.
  • the normal saline group (Cohort 1) showed a bodyweight loss within 20%, mainly due to the leukemia burden.
  • Q3Dx6 dose group (Cohort 2), 4 of 5 mice tolerated the NDC well ( ⁇ 20% loss), and bodyweight was gained after 6 doses; while the remaining mouse showed >20% bodyweight loss after the 5 th dose, and more bodyweight loss after the 6 th dose.
  • Q3Dx3 0.50 mg/kg
  • all 5 mice tolerated the NDC well ( ⁇ 20% loss), and bodyweight was gained after 3 doses.
  • FIG. 28 provides the in vivo bioluminescence images (BLI) obtained from the AML mice treated with normal saline or the exemplary NDC at each dose regimen (i.e ., Cohorts 1-4 from Table 6).
  • Quantitative in vivo bioluminescence imaging analysis of Cohorts 1-4 i.e., AML mice treated with normal saline or the exemplary NDC at each dose regimen outlined in Table 6) is provided in FIG. 29 .
  • the leukemia burden continued to progress, with the average whole-body BLI signal increasing >90 fold in 34 days, while a quick and dose-dependent suppression of the leukemia burden was achieved in all 3 treatment groups (Cohorts 2-4).
  • the 0.5 mg/kg (Q3Dx3) dose group (Cohort 3) showed 11-fold less leukemia burden on Day 34 when compared with burden on Day 1 post-leukemia injection.
  • 0.33 mg/kg (Q3Dx6) dose group (Cohort 2) with the 0.65 mg/kg (Q3Dx3) dose group (Cohort 4)
  • 0.33 mg/kg was tolerated better with a slightly better response.
  • FIG. 30 provides a graph illustrating the results of bone marrow aspiration of Cohorts 1-4 (i.e., the mice treated with normal saline or the exemplary NDC at each dose group indicated in Table 6) on Day 42 post-leukemia injection.
  • Leukemia was detected in the group of mice treated with normal saline (Cohort 1), while no detectable leukemia burden could be observed in any of the mice from the treatment groups (Cohorts 2-4).
  • the NDCs were incubated in 0.9% saline, PBS, human plasma (10%), and mouse plasma (10%) at 37 °C in a shaking dry bath for different time periods.
  • plasma proteins in the samples were removed by precipitation, through addition of an equivalent volume of cold acetonitrile, followed by centrifugation at 10000 rpm in an Eppendorf 5425 microcentrifuge. Following centrifugation, the clear supernatant was transferred from the centrifuge tube into a clear total recovery HPLC vial. The supernatant free of any visible aggregation was diluted with an equivalent volume of deionized water to adjust the sample matrix to match the starting conditions of the HPLC separation and avoid loss of sensitivity. The purity and impurity of each sample is then quantified by RP-HPLC.
  • the targeted-NDCs produced using the methods described in Example 3, using the diene-silane precursor exhibited high stability in mouse and human plasma, and showed significant stability improvement, relative to corresponding NDCs produced using an amine-silane precursor (see FIGS. 33A and 33B )).
  • the NDCs prepared using the diene-silane precursor more than 95% of exatecan drugs remain on the NDCs for up to 7 days in mouse and human plasma, obtained by the UV-Vis spectra of the NDC peaks in RP-HPLC chromatograms.
  • RP-HPLC assay monitoring free exatecan suggested that the released exatecan was below detection limit of RP-HPLC, i.e., 0.02%, and the absence of non-desired free drug further demonstrates their high plasma stability.
  • the targeted-NDCs also exhibited high storage stability at 4 °C in 0.9% saline. Their purity, size distribution, and hydrodynamic diameter were characterized by RP-HPLC, SEC and FCS respectively, and remained unchanged over 6 months under storage condition. Such high storage stability is another key parameter important for both clinical translation and commercial manufacture.
  • an exemplary NDC The pharmacokinetics and toxicology of an exemplary NDC were assessed in a rat model and in a dog model.
  • the NDC used in this study was prepared according to Example 3, using the exatecan-linker conjugate precursor compound 202 of Example 1.
  • this exemplary NDC is highly stable in plasma and elicits antitumor efficacy in a variety of cell line and PDX-derived tumor models both in vitro and in vivo.
  • TK parameters estimated in the 15-day GLP study, revealed similar plasma exposure values in males and females for the NDC, total exatecan (conjugated and released) and released exatecan.
  • the NDC exhibited an average circulatory half-life ranging from approximately 15 to 20 hours in rats, and 24 to 29 hours in dogs, with no accumulation of the NDC, total exatecan, or free exatecan observed from day 1 to day 15.
  • Based upon AUC 0-last (hr*ng/mL) released payload levels in the circulation were less than approximately 0.3% and 0.1% of the total payload levels in the rat and the dog respectively.
  • No NDC anti-drug antibodies were induced in either species.
  • the NDC has a favorable nonclinical safety/TK profile.

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